Methods and compositions for inducing hygienic behavior in honey bees

ABSTRACT

The presently disclosed subject matter provides tritriacontene compositions for inducing hygienic behavior in honey bees; mite-infested brood extract compositions for inducing hygienic behavior in honey bees; methods of inducing hygienic behavior in honey bees; methods of selecting one or more honey bee(s) exhibiting hygienic behavior, and methods for assessing the degree of hygienic behavior within a honey bee colony.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent application Ser. No. 15/742,072 filed Jan. 5, 2018, which is a 35 U.S.C. § 371 national phase application of PCT Application PCT/US16/40993 filed Jul. 5, 2016, which claims priority to U.S. Provisional Patent Application No. 62/188,991 filed on Jul. 6, 2015. The contents of each are hereby incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. 2010-65104-20533 awarded by the National Institute of Food and Agriculture, an agency within the United States Department of Agriculture. The government has certain rights in the invention.

FIELD

The inventions relate to the fields of agriculture, apiculture and hygienic behavior in honey bees.

BACKGROUND

Honey bees are the most important commercial pollinator world-wide for agricultural production and food security. Agriculture depends on animal pollination for over 100 different crops that provide >30% of the human diet and also supply fiber, fuel, and drugs. [3, 4] Honey bees are responsible for 80% of managed insect pollination and their economic impact on U.S. food crops in the year 2000 has been estimated to be $14.6 billion. [3] However, honey bees and other insect pollinators suffer from a combination of factors, including habitat loss, pesticide exposure, pathogens, and stress due to active management. [15, 16] Recently, honey bee health has been marked with sharp declines and losses of colonies, with reports of “colony collapse disorder.” [17] The failure to identify a single factor as the cause of colony collapse disorder indicates that the global honey bee health crisis is complex and heterogeneous: Multiple stressors may act synergistically to lead to symptomatic health declines and colony failure [18] and the causes may vary in time and between locations. [19]

While no single factor has been identified as the cause of colony collapse disorder, the introduction and spread of parasites and associated pathogens and pesticide exposure have a central role in the health decline of honey bees. [17, 20-29] Disease control in the honey bee is crucial because it remains the most important commercial pollinator world-wide and its pollination services are irreplaceable in many agricultural systems. Moreover, honey bees are significant pollinators in natural ecosystems and are potential vectors of diseases that threaten native pollinator communities. [6]

The ectoparasitic mite Varroa destructor in particular is a threat to honey bee health and apiculture today. [30, 31, 33, 41, 42] Varroa enter honey bee colonies to complete its reproductive cycle on developing honey bee brood. [30, 31] Mature, fertilized Varroa females enter a brood cell to lay eggs, and both the Varroa female and its offspring feed on the brood. [35] Varroa causes physical and physiological damage when feeding on the brood. Varroa is a vector of viruses and has been associated with viral amplification and honey bee disease susceptibility. Miticides used to control Varroa infestations are problematic because of toxicity to honey bees, beekeepers, and crops; general ecosystem pollution; and resistance development in Varroa.

Varroa is conventionally treated with synthetic acaricides, most notably coumaphos, tau-fluvalinate, flumethrin, and amitraz. [31] These substances are toxic and persistent and may accumulate in the hive [64], consequently harming honey bee health. [65, 66] For example, miticides fluvalinate and coumaphos, have been found to have lethal and sublethal effects on honey bee queens, workers, and drones. [2, 31, 64-66, 132, 154, 162, 177, 181] Moreover, resistance build-up in resident Varroa populations decreases the efficacy of chemical control. [31, 72, 178] Synergistic effects of fluvalinate and coumaphos have been measured, where the toxicity of each chemical is significantly increased in bees previously exposed to the other. [172] Furthermore, immunosuppression caused by chemical exposure makes honey bees more susceptible to parasites like Varroa, as well as to the pathogens they vector. [64, 70, 181, 184] In addition to affecting honey bee health, miticides compromise beekeeper health [158], enter bee products including those consumed by humans [64, 157, 174, 185] and contribute to general ecosystem pollution.

Other control strategies such as physical mite removal and use of organic acids and essential oils have been proposed as alternatives, but have many limitations that compromise efficacy, such as the labor-intensive application, temperature-sensitivity, and potential side-effects on honey bees. [74, 75, 171] Despite substantial evidence of the need, no adequate solution for control of Varroa has been developed. [75]

Traditional efforts to keep honey bees healthy have focused on management techniques and treatments (primarily chemotherapies). These treatments may lead to a loss of honey, are expensive and labor-intensive, and pose human health risks. [62, 63] Moreover, their long-term efficacy is questionable: None of the honey bee diseases have been eradicated due to past management. On the contrary, a steady emergence of novel pests and pathogens can be observed. [19]

Beekeepers have selectively bred honey bees for hygienic behavior as an alternative. [78, 87, 92, 155, 183] The mechanism for hygienic behavior is not completely understood, but the trait can be measured, for instance, by frequency of certain behaviors in honey bees. Minnesota Hygienic (HYG) is a breed of honey bees based on the high frequency of honey bee removal of freeze-killed brood. [83] A circle of brood is frozen with liquid nitrogen and percent removal of the killed brood is recorded, thus assessing the effectiveness of the general detection and removal of dead brood. [83] However, the olfactory trigger for hygienic removal of mite-infested and other live brood may be significantly lower than that of dead brood. [10, 100, 112, 156, 175, 183] HYG breeding relies on olfactory triggers of dead brood and results in bred honey bees that may lack sufficient sensitivity to living diseased brood. [94, 100] Another example is Varroa Sensitive Hygienic (VSH), which is a breed of honey bees based on measured changes in mite reproduction. [85] VSH breeding is based on a more narrowly defined goal but hygienic behavior may just be one mechanism for these bees to suppress mite reproduction. It has been unclear whether VSH bees are truly distinct or whether suppression of mite reproduction is due to the interruption of the mite reproductive cycle by hygienic removal of infested brood. [31, 84, 85, 170, 196] While the HYG and VSH hygienic honey bee colonies exhibit reduced mite and disease loads compared to unselected hives [9, 84, 167, 182], the selective breeding programs are not widely adopted by beekeepers due to lack of specificity, difficulty and expense of current selection assays. [92-95, 99, 183, 197] Also, hygienic lines do not yet serve as complete alternatives to chemical Varroa control [75], as chemical treatments are sometimes required to control severe mite infestations in hygienic hives. [170, 182]

Moreover, despite the presence of natural honey bee social immune mechanisms like hygienic behavior, honey bee health is currently being severely threatened. Following global pollinator population trends [2, 189], managed honey bee colonies in the United States have declined steadily for over six decades, from 5.9 million colonies in 1947 to 2.4 million colonies in 2005 [190, 191]. Total annual colony losses in the United States have exceeded 33% in four of the last five years, exceeding 45% between April 2012 and April 2013. [192, 193, 194, 195] Honey bee losses are largely attributed to introduction and spread of new parasites and associated pathogens, and to lethal and sublethal effects of agrochemical exposure. [2, 47, 195]

The long-term sustainability of apiculture depends on the balance between the benefit of honey bee keeping to the individual and the costs of honey bee management and losses. There remains a need for improving selection of honey bees for hygienic behavior and use of hygienic behavior by honey bees to prevent or treat a diseased honey bee colony to ensure adequate supplies of managed pollinators for agriculture.

SUMMARY

The presently disclosed subject matter provides tritriacontene compositions for inducing hygienic behavior in honey bees; mite-infested brood extract compositions for inducing hygienic behavior in honey bees; methods of inducing hygienic behavior in honey bees; methods of selecting one or more honey bee(s) exhibiting hygienic behavior, and methods for assessing the degree of hygienic behavior within a honey bee colony.

In some embodiments, the presently disclosed subject matter is directed to a composition for inducing hygienic behavior in honey bees comprising a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees. In a further embodiment, the tritriacontene is a stereoisomer, racemic mixture or optically active mixture. The various stereoisomers include geometric isomers/diastereomers (e.g. cis-isomers and trans-isomers, also referred to as Z-isomers and E-isomers) and enantiomers; and refers to isomers that differ only in the way the atoms are arranged in space. In one embodiment, the tritriacontene is a trans-isomer. In one embodiment, the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof. In one embodiment, the tritriacontene is a cis-isomer. In one embodiment, the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof.

In some embodiments, the presently disclosed subject matter is directed to a composition for inducing hygienic behavior in honey bees comprising mite-infested brood extract and an agriculturally acceptable diluent or carrier, wherein the mite-infested brood extract is in an effective amount for inducing hygienic behavior in honey bees. In one embodiment, the mite-infested brood extract has a concentration of 3, 1, or 0.3 brood equivalents.

In other embodiments, the presently disclosed subject matter is directed to a method of inducing hygienic behavior in honey bees, the method comprising contacting hive cells with a composition comprising a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees. In a further embodiment, the tritriacontene is a stereoisomer, racemic mixture or optically active mixture. The various stereoisomers include geometric isomers/diastereomers (e.g. cis-isomers and trans-isomers, also referred to as Z-isomers and E-isomers) and enantiomers; and refers to isomers that differ only in the way the atoms are arranged in space. In one embodiment, the tritriacontene is a trans-isomer. In one embodiment, the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof. In one embodiment, the tritriacontene is a cis-isomer. In one embodiment, the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof.

In other embodiments, the presently disclosed subject matter is directed to a method of inducing hygienic behavior in honey bees, the method comprising contacting hive cells with a composition comprising a mite-infested brood extract and an agriculturally acceptable diluent or carrier, wherein the mite-infested brood extract is in an effective amount for inducing hygienic behavior in honey bees. In one embodiment, the mite-infested brood extract has a concentration of 3, 1, or 0.3 brood equivalents.

In another embodiment, the hygienic behavior comprises eating diseased brood or diseased honeybees, removing diseased brood or diseased honeybees from hive cells, removing pests or parasites, uncapping hive cells, or uncapping and recapping hive cells. In a further embodiment, the diseased brood or diseased honeybees are infested with pests or parasites; infected with a pathogen; or damaged. In a further embodiment, the diseased brood or diseased honeybees are infested with mites; more particularly, wherein the mites are mites of the genus Varroa; particularly wherein the mites are mites of the species Varroa destructor or Varroa jacobsoni. In another embodiment, the hygienic behavior results in survival of a honey bee colony. In another embodiment, the hygienic behavior results in suppression of mite reproduction, decreased mite survival, or suppression of a mite infestation. In another embodiment, the hive cells are capped hive cells or uncapped hive cells. In another embodiment, the contacting of the hive cells is on one or more days after the hive cells are capped. In another embodiment, the contacting of the hive cells is on one or more days before the hive cells are capped. In another embodiment, the hive cells are worker-brood cells, drone-brood cells, or queen bee cells. In yet another embodiment the diseased brood are eggs, larvae, or pupae.

In additional embodiments, the presently disclosed subject matter is directed to a method for selecting one or more honey bee(s) exhibiting hygienic behavior comprising a) applying a tritriacontene composition to a set of hive cells; b) performing an assay to identify a hygienic colony, wherein the assay comprises exposing the set of hive cells to a test colony; and c) selecting one or more honey bee(s) from an identified hygienic colony, wherein the selected one or more honey bee(s) exhibit hygienic behavior. In further embodiments, the set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the tritriacontene composition comprises a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees. In a further embodiment, the tritriacontene is a stereoisomer, racemic mixture or optically active mixture. The various stereoisomers include geometric isomers/diastereomers (e.g. cis-isomers and trans-isomers, also referred to as Z-isomers and E-isomers) and enantiomers; and refers to isomers that differ only in the way the atoms are arranged in space. In one embodiment, the tritriacontene is a trans-isomer. In one embodiment, the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof. In one embodiment, the tritriacontene is a cis-isomer. In one embodiment, the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof.

In additional embodiments, the presently disclosed subject matter is directed to a method for selecting one or more honey bee(s) exhibiting hygienic behavior comprising a) applying a mite-infested brood extract composition to a set of hive cells; b) performing an assay to identify a hygienic colony, wherein the assay comprises exposing the set of hive cells to a test colony; and c) selecting one or more honey bee(s) from an identified hygienic colony, wherein the selected one or more honey bee(s) exhibit hygienic behavior. In further embodiments, the set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the mite-infested brood extract composition comprises a mite-infested brood extract and an agriculturally acceptable diluent or carrier, wherein the mite-infested brood extract is in an effective amount for inducing hygienic behavior in honey bees. In one embodiment, the mite-infested brood extract has a concentration of 3, 1, or 0.3 brood equivalents.

In another embodiment, each of the selected honey bee(s) is a queen bee or drone bee. In another embodiment, the assay further comprises i) determining the amount of emptied hive cells in the set of hive cells; and ii) identifying a hygienic colony, wherein a test colony is a hygienic colony if at least 90% of the set of hive cells are emptied; more particularly wherein the emptied hive cells have no eggs, larvae, or pupae, or contain partially eaten larvae or pupae; particularly wherein the emptied hive cells are capped hive cells or uncapped hive cells. In another embodiment, the emptied hive cells have no diseased honey bees, or contain partially eaten diseased honey bees. In another embodiment, the hive cells are capped hive cells and the assay further comprises i) determining the amount of uncapped and/or recapped hive cells in the set of capped hive cells; and ii) identifying a hygienic colony, wherein a test colony is a hygienic colony if at least 90% of the set of capped hive cells are uncapped and/or recapped.

In another embodiment, the method further comprises d) mating a selected honey bee with one or more honey bee(s) from at least one separately identified hygienic colony to produce offspring. In another embodiment, the method further comprises e) raising the offspring, f) applying a tritriacontene composition to a second set of hive cells, g) performing a second assay to identify whether the raised offspring is a hygienic colony, wherein the second assay comprises exposing the second set of hive cells to the raised offspring, and h) selecting one or more honey bee(s) from an identified hygienic colony, wherein the selected one or more honey bee(s) exhibit hygienic behavior. In further embodiments, the second set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the tritriacontene composition comprises a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees. In a further embodiment, the tritriacontene is a stereoisomer, racemic mixture or optically active mixture. The various stereoisomers include geometric isomers/diastereomers (e.g. cis-isomers and trans-isomers, also referred to as Z-isomers and E-isomers) and enantiomers; and refers to isomers that differ only in the way the atoms are arranged in space. In one embodiment, the tritriacontene is a trans-isomer. In one embodiment, the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof. In one embodiment, the tritriacontene is a cis-isomer. In one embodiment, the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof.

In another embodiment, the method further comprises d) mating a selected honey bee with one or more honey bee(s) from at least one separately identified hygienic colony to produce offspring. In another embodiment, the method further comprises e) raising the offspring, f) applying a mite-infested brood extract composition to a second set of hive cells, g) performing a second assay to identify whether the raised offspring is a hygienic colony, wherein the second assay comprises exposing the second set of hive cells to the raised offspring, and h) selecting one or more honey bee(s) from an identified hygienic colony, wherein the selected one or more honey bee(s) exhibit hygienic behavior. In further embodiments, the second set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the mite-infested brood extract composition comprises a mite-infested brood extract and an agriculturally acceptable diluent or carrier, wherein the mite-infested brood extract is in an effective amount for inducing hygienic behavior in honey bees. In one embodiment, the mite-infested brood extract has a concentration of 3, 1, or 0.3 brood equivalents.

In another embodiment, the second assay further comprises i) determining the amount of emptied hive cells in the second set of hive cells; and ii) identifying a hygienic colony, wherein the raised offspring is a hygienic colony if at least 90% of the second set of hive cells are emptied. In another embodiment, the emptied hive cells are capped hive cells or uncapped hive cells. In another embodiment, the hive cells are capped hive cells and the second assay further comprises i) determining the amount of uncapped and/or recapped hive cells in the second set of capped hive cells; and ii) identifying a hygienic colony, wherein the raised offspring is a hygienic colony if at least 90% of the second set of capped hive cells are uncapped and/or recapped.

In some embodiments, the presently disclosed subject matter is directed to a method for assessing the degree of hygienic behavior within a honey bee colony comprising a) applying a tritriacontene composition to a set of hive cells; b) exposing the set of hive cells to a honey bee colony; and c) determining the amount of emptied hive cells in the set of hive cells; wherein a higher amount of the set of hive cells that are emptied is associated with a greater degree of hygienic behavior. In another embodiment, the emptied hive cells are capped hive cells or uncapped hive cells. In further embodiments, the set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the tritriacontene composition comprises a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees. In a further embodiment, the tritriacontene is a stereoisomer, racemic mixture or optically active mixture. The various stereoisomers include geometric isomersidiastereomers (e.g. cis-isomers and trans-isomers, also referred to as Z-isomers and E-isomers) and enantiomers; and refers to isomers that differ only in the way the atoms are arranged in space. In one embodiment, the tritriacontene is a trans-isomer. In one embodiment, the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof. In one embodiment, the tritriacontene is a cis-isomer. In one embodiment, the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof.

In some embodiments, the presently disclosed subject matter is directed to a method for assessing the degree of hygienic behavior within a honey bee colony comprising a) applying a mite-infested brood extract composition to a set of hive cells; b) exposing the set of hive cells to a honey bee colony; and c) determining the amount of emptied hive cells in the set of hive cells; wherein a higher amount of the set of hive cells that are emptied is associated with a greater degree of hygienic behavior. In another embodiment, the emptied hive cells are capped hive cells or uncapped hive cells. In further embodiments, the set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the mite-infested brood extract composition comprises a mite-infested brood extract and an agriculturally acceptable diluent or carrier, wherein the mite-infested brood extract is in an effective amount for inducing hygienic behavior in honey bees. In one embodiment, the mite-infested brood extract has a concentration of 3, 1, or 0.3 brood equivalents.

In other embodiments, the presently disclosed subject matter is directed to a method for assessing the degree of hygienic behavior within a honey bee colony comprising a) applying a tritriacontene composition to a set of capped hive cells; b) exposing the set of capped hive cells to a honey bee colony; and c) determining the amount of uncapped and/or recapped hive cells in the set of capped hive cells; wherein a higher amount of the set of hive cells that are uncapped and/or recapped is associated with a greater degree of hygienic behavior. In further embodiments, the set of capped hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the set of capped hive cells is empty. In another embodiment, the tritriacontene composition comprises a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees. In a further embodiment, the tritriacontene is a stereoisomer, racemic mixture or optically active mixture. The various stereoisomers include geometric isomers/diastereomers (e.g. cis-isomers and trans-isomers, also referred to as Z-isomers and E-isomers) and enantiomers; and refers to isomers that differ only in the way the atoms are arranged in space. In one embodiment, the tritriacontene is a trans-isomer. In one embodiment, the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof. In one embodiment, the tritriacontene is a cis-isomer. In one embodiment, the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof.

In other embodiments, the presently disclosed subject matter is directed to a method for assessing the degree of hygienic behavior within a honey bee colony comprising a) applying a mite-infested brood extract composition to a set of capped hive cells; b) exposing the set of capped hive cells to a honey bee colony; and c) determining the amount of uncapped and/or recapped hive cells in the set of capped hive cells; wherein a higher amount of the set of hive cells that are uncapped and/or recapped is associated with a greater degree of hygienic behavior. In further embodiments, the set of capped hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the set of capped hive cells is empty. In another embodiment, the mite-infested brood extract composition comprises a mite-infested brood extract and an agriculturally acceptable diluent or carrier, wherein the mite-infested brood extract is in an effective amount for inducing hygienic behavior in honey bees.

Certain aspects of the presently disclosed subject matter having been stated above, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

FIG. 1 shows 2013 and 2014 data for treatment, hive type and brood type effects on mean percent brood removal. White, gray and black bars represent control (CON), HYG and VSH brood, respectively. Different letters indicate significant differences from Chi-square analysis after Bonferroni correction (p<0.0167) within each Treatment by Hive Type by Year combination.

FIG. 2 shows treatment effects on mean percent brood removal. Different letters indicate significant differences from Chi-square analysis (p<0.05).

FIG. 3 shows hive type effects on mean percent brood removal. Different letters indicate significant differences from Chi-square analysis (p<0.05).

FIG. 4 shows hive type effects on mean percent brood removal for the subset of data in which hive type was the same as brood type. Different letters indicate significant differences from Chi-square analysis (p<0.05).

FIG. 5 shows brood type effects on mean percent brood removal. Different letters indicate significant differences from Chi-square analysis (p<0.05).

FIG. 6A shows average percent brood removal plotted against the level of hygiene of the brood's hive of origin. White, gray and black symbols represent control (CON), HYG and VSH brood, respectively. Circular, square and triangular markers represent control, wound and mite treatments, and are represented by lines of best fit C, B and A, respectively.

FIG. 6B shows average percent brood removal plotted against the level of hygiene of the brood's host hive. White, gray and black symbols represent control (CON), HYG and VSH hives, respectively. Circular, square and triangular markers represent control, wound and mite treatments, and are represented by lines of best fit C, B and A, respectively.

FIG. 7 shows the GCMS output from a single, mite-infested honey bee, illustrating the location of the P32 peak (top) and the mass-to-charge ratio for P32 (bottom).

FIG. 8 shows the relative quantity of peak number 32 (P32) (a tritriacontene) in a control treatment brood subset versus a Varroa mite-treated brood subset in three types of brood (control (CON) Brood, HYG Brood, and VSH brood), based on averages of samples of each type of brood (top row); and the relative quantity of P32 in a brood subset with low levels of Deformed Wing Virus versus a brood subset with high levels of Deformed Wing Virus in three types of brood (control (CON) Brood. HYG Brood, and VSH Brood), based on averages of samples of each type of brood. (bottom row). All relative quantities of P32 are based on brood extract analysis using Gas Chromotography Mass Spectrometry (GCMS). From ANOVAs: p=0.031 for VSH mite-treated brood subset vs. VSH control brood subset; p=0.014 for Control brood subset with high levels of Deformed Wing Virus vs. Control brood subset with low levels of Deformed Wing Virus; p=0.001 for HYG brood subset with high levels of Deformed Wing Virus vs. Control brood subset with low levels of Deformed Wing Virus.

FIG. 9A shows a graphic depicting a set of hive cells with Varroa-infested brood (dark shading) and control brood (medium shading); an image depicting a fine-tipped paint brush introducing Varroa to an opened hive cell that had been recently capped; a graphic depicting an uncapped hive cell with Varroa-infested brood, a capped hive cell with Varroa-infested brood, and a capped hive cell with control brood; and a graphic depicting the Gas Chromatography-Mass Spectrometry (GCMS) setup to determine relative P32 quantities from the brood extracts.

FIG. 9B shows the relative quantity of P32 of the brood extract from uncapped hive cells with Varroa-infested brood, the brood extract from capped hive cells with Varroa-infested brood, and the brood extract from capped hive cells with control brood. Each relative quantity of P32 is based on an average of six samples for each cell type. Different letters indicate statistically significant differences (p<0.05) as determined from a paired-sample t-test.

FIG. 9C shows the relative quantity of P32 quantity of the brood extract from uncapped hive cells with Varroa-infested brood, the brood extract from capped hive cells with Varroa-infested brood, and the brood extract from capped hive cells with control brood. For each mean, 95% CI intervals are provided. Different letters indicate a statistically significant difference (p<0.0167) in mean P32 quantity between cell types as determine by ANOVA. Each relative quantity of P32 is based on an average of 24 samples for each cell type.

FIG. 10 shows the mean percent of brood cells uncapped 8 hours after no treatment (none), treatment with hexane, treatment with control (not mite-infested) brood extract (at three concentrations: 3, 1 or 0.3 brood equivalents (BEqs)), and treatment with mite-infested brood extract (at three concentrations: 3, 1 or 0.3 BEqs). Mean percent of brood cells uncapped is based on a sample size of 30 cells each for no treatment and hexane treatment, and 15 cells for each brood extract concentration.

FIG. 11 shows the mean percent of brood cells uncapped 8 hours after treatment with control (not mite-infested) brood extract from control and treatment with mite-infested brood extract. Each bar represents a combination of data from like treatments for all three brood extract concentrations as shown in FIG. 10. Thus each bar is based on a sample size of 45 cells. Different letters indicate a statistically significant difference (p<0.05) as determined from a Chi-square test.

FIG. 12 shows the percent uncapping and removal of brood underneath treated wax caps after 24 hours. Using the airbrushing method, approximately 100l of solvent were applied to each chemically treated cell. Different letters indicate a statistically significant difference (p<0.0018) as determined from Chi-square tests with Bonferroni correction.

FIG. 13 shows a significant positive correlation between the complete removal of brood treated with P32 at 24 hours and brood treated with liquid nitrogen (FKB assay).

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the presently disclosed subject matter are shown. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Preferred methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

Throughout the present specification, numerical ranges are provided for certain quantities. It is to be understood that these ranges comprise all subranges therein. Thus, the range “from 50 to 80” includes all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.). Furthermore, all values within a given range may be an endpoint for the range encompassed thereby (e.g., the range 50-80 includes the ranges with endpoints such as 55-80, 50-75, etc.).

The presently disclosed subject matter provides tritriacontene compositions for inducing hygienic behavior in honey bees; mite-infested brood extract compositions for inducing hygienic behavior in honey bees; methods of inducing hygienic behavior in honey bees; methods of selecting one or more honey bee(s) exhibiting hygienic behavior, and methods for assessing the degree of hygienic behavior within a honey bee colony. As described more fully below, the presently disclosed subject matter relates to the finding that a tritriacontene compound triggers hygienic behavior in honey bees

I. COMPOSITIONS

In some embodiments, the presently disclosed subject matter is directed to a composition for inducing hygienic behavior in honey bees comprising a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees. As used herein, the term “tritriacontene” refers to a compound with the molecular formula C₃₃H₆₆. In a further embodiment, the tritriacontene is a stereoisomer, racemic mixture or optically active mixture. The various stereoisomers include geometric isomers/diastereomers (e.g. cis-isomers and trans-isomers, also referred to as Z-isomers and E-isomers) and enantiomers; and refers to isomers that differ only in the way the atoms are arranged in space. In one embodiment, the tritriacontene is a trans-isomer. In one embodiment, the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof. In one embodiment, the tritriacontene is a cis-isomer. In one embodiment, the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof. It will be appreciated by those skilled in the art that one stereoisomer may be more active than the others. Individual stereoisomers and optically active mixtures may be obtained by selective synthetic procedures, by conventional synthetic procedures using resolved starting materials, or by conventional resolution procedures.

As used herein, the term “agriculturally acceptable derivative” refers to any agriculturally acceptable salt, ester, or salt of such ester, of such compound, or any other adduct or derivative which, upon administration, is capable of providing (directly or indirectly) a compound as otherwise described herein, or residue thereof. Materials and methods for derivatizing the parent compounds are known and may be adapted to the present invention. Certain exemplary pharmaceutical compositions and pharmaceutically acceptable derivatives will be discussed in more detail herein below.

As used herein, the term “agriculturally acceptable salt,” refers to salts of a free acid or a free base which are not biologically undesirable and are generally prepared by reacting the free base with a suitable organic or inorganic acid or by reacting the acid with a suitable organic or inorganic base. Agriculturally acceptable salts include salts, the cations or anions of which are known in the art for the formation of salts for agricultural or apicultural use. In some embodiments the salts are water-soluble. Suitable cations include the ions of the alkali metals (such as lithium, sodium and potassium), alkaline earth metals (such as calcium, magnesium and barium) and transition metals (such as manganese, copper, zinc and iron), and amines. Suitable anions of acid addition salts are primarily chloride, bromide, fluoride, hydrogen sulfate, sulfate, dihydrogen phosphate, hydrogen phosphate, phosphate, nitrate, hydrogen carbonate, carbonate, hexafluorosilicate, hexafluorophosphate, benzoate, and the anions of C₁-C₄-alkanoic acids, such as formate, acetate, propionate and butyrate. The term “agriculturally acceptable ester” refers to those that are or can by hydrolyzed, oxidized, metabolized, or otherwise converted, e.g., in plants, water, or soil, to the corresponding carboxylic acid which, depending on the pH, may be in the dissociated or un-dissociated form. Exemplary esters include those derived from C₁-C₁₂ alkyl, C₃-C₁₂ alkenyl, C₃-C₁₂ alkynyl or C₇-C₁₀ aryl-substituted alkyl alcohols. Esters can be prepared by coupling of the acid with the alcohol using any number of suitable activating agents such as those used for peptide couplings such as dicyclohexylcarbodiimide (DCC) or carbonyl diimidazole (CDI); by reacting the acid with alkylating agents such as alkylhalides or alkylsulfonates in the presence of a base such as triethylamine or lithium carbonate; by reacting the corresponding acid chloride of an acid with an appropriate alcohol; by reacting the corresponding acid with an appropriate alcohol in the presence of an acid catalyst or by transesterification.

In some embodiments, the tritriacontene can form stable complexes with solvent molecules that remain intact after the non-complexed solvent molecules are removed from the compound. These complexes are often referred to as “solvates.”

In some embodiments, the presently disclosed subject matter is directed to a composition for inducing hygienic behavior in honey bees comprising a mite-infested brood extract and an agriculturally acceptable diluent or carrier, wherein the mite-infested brood extract is in an effective amount for inducing hygienic behavior in honey bees. As used herein, the term “mite-infested brood extract” refers to extract derived from mite-infested honey bee brood and comprising cuticular chemicals; or extract derived from honey bee brood and comprising cuticular chemicals and artificially added tritriacontene. In one embodiment, the mite-infested brood extract has a concentration of 3, 1, or 0.3 brood equivalents. Mite-infested brood extract may be derived from an individual brood; multiple brood; whole or part of individual brood(s); and living, dead, or damaged brood. Mite-infested brood extract may be obtained using conventional methods, including, but not limited to solvent extraction. Any of a variety of solvents including polar solvents, non-polar solvents, or combinations of two or more thereof may be used in solvent extraction. Suitable non-polar solvents include non-polar organic solvents such as alkanes, including C₁-C₈ alkanes, cycloalkanes, including C₁-C₈ alkanes, alkyl ethers, including C₁-C₈ alkyl ethers, Petroleum ethers, ketones, including C₁-C₈ ketones, methylene chloride, ethyl acetate, xylene, toluene, chloroform, vegetable oil, mineral oil and the like. In one embodiment, the mite-infested brood extract is obtained from mite-infested honey bee brood by solvent extraction using a hexane solvent. Suitable polar solvents include polar inorganic solvents such as water and the like, polar organic solvents such as alcohols and corresponding organic acids, for example C₁-C₈ alcohols including methanol, ethanol, propanol, butanol, and the like and organic acids, including acetic acid, formic acid, propanoic acid, and the like, polyols and glycols, including C₁-C₈ polyols/glycols and the like, and combinations of two or more thereof.

As used herein, the term “agriculturally acceptable diluent or carrier,” refers to organic or inorganic material, natural or synthetic, that facilitates application and is agriculturally or apiculturally acceptable on the application surface, such as, adjuvants, mixers, enhancers, or combinations thereof suitable for application of the composition. Examples of suitable liquid agriculturally acceptable carriers include hexane, pentane, water, toluene, xylene, petroleum naphtha, crop oil, acetone, methyl ethyl ketone, cyclohexanone, trichloroethylene, perchloroethylene, ethyl acetate, amyl acetate, butyl acetate, propylene glycol monomethyl ether and diethylene glycol monomethyl ether, methanol, ethanol, isopropanol, amyl alcohol, ethylene glycol, propylene glycol, and glycerine. Exemplary solid agriculturally acceptable carriers include talc, pyrophyllite clay, silica, attapulgus clay, kieselguhr, chalk, diatomaceous earth, lime, calcium carbonate, bentonite clay, Fuller's earth, cotton seed hulls, wheat flour, soybean flour, pumice, wood flour, walnut shell flour, and lignin. Additional adjuvants include antifoam agents, neutralizing agents, buffers, dispersing agents, thickening agents, sequestering agents, and so on.

The presently disclosed subject matter contemplates all vehicles by which the composition of the presently disclosed subject matter can be formulated for delivery and use as solutions, suspensions, emulsions, wettable powders and water dispersible granules, dry flowables, emulsifiable concentrates, granules, dusts, fumigants, gels, microencapsulations, and the like. The compositions can be manufactured in a manner known in the art; for example, without limiting the foregoing, by means of conventional mixing, dissolving, granulating, or emulsifying processes.

Formulations for application to hive cells may be applied following dilution of the concentrated formulation with water as aqueous solutions, suspensions or emulsions, or combinations thereof. Such solutions, suspensions or emulsions are produced from water-soluble, water-suspended or water-suspendable, water-emulsified or water-emulsifiable formulations or combinations thereof which are solids, including wettable powders or water dispersible granules, or liquids including emulsifiable concentrates, aqueous suspensions or suspension concentrates, and aqueous emulsions or emulsions in water, or mixtures thereof such as suspension-emulsions.

Wettable powders, which may be compacted to form water dispersible granules, comprise a mixture of the active ingredient, an inert carrier, and surfactants. The concentration of the active ingredient in the wettable powder is usually from about 10 percent to about 90 percent by weight based on the total weight of the wettable powder, more preferably about 25 weight percent to about 75 weight percent. In the preparation of wettable powder formulations, the active ingredients can be compounded with any finely divided solid, such as prophyllite, talc, chalk, gypsum, Fuller's earth, bentonite, attapulgite, starch, casein, gluten, montmorillonite clays, diatomaceous earths, purified silicates or the like. In such operations, the finely divided carrier and surfactants are typically blended with the compound(s) and milled.

Emulsifiable concentrates of the active ingredient comprise a concentration, such as from about 10 weight percent to about 50 weight percent of the active ingredient, in a suitable liquid, based on the total weight of the concentrate. The active ingredients are dissolved in an inert carrier, which is either a water miscible solvent or a mixture of water-immiscible organic solvents, and emulsifiers. The concentrates may be diluted with water and oil to form spray mixtures in the form of oil-in-water emulsions. Useful organic solvents include aromatics, especially the high-boiling naphthalenic and olefinic portions of petroleum such as heavy aromatic naphtha. Other organic solvents may also be used, such as, for example, terpenic solvents, including rosin derivatives, aliphatic ketones, such as cyclohexanone, and complex alcohols, such as 2-ethoxyethanol.

Emulsifiers which can be advantageously employed herein can be readily determined by those skilled in the art and include various nonionic, anionic, cationic and amphoteric emulsifiers, or a blend of two or more emulsifiers. Examples of nonionic emulsifiers useful in preparing the emulsifiable concentrates include the polyalkylene glycol ethers and condensation products of alkyl and aryl phenols, aliphatic alcohols, aliphatic amines or fatty acids with ethylene oxide, propylene oxides such as the ethoxylated alkyl phenols and carboxylic esters esterified with the polyol or polyoxyalkylene. Cationic emulsifiers include quaternary ammonium compounds and fatty amine salts. Anionic emulsifiers include the oil-soluble salts (e.g., calcium) of alkylaryl sulfonic acids, oil-soluble salts of sulfated polyglycol ethers and appropriate salts of phosphated polyglycol ether.

Representative organic liquids which can be employed in preparing emulsifiable concentrates are the aromatic liquids such as xylene, propyl benzene fractions; or mixed naphthalene fractions, mineral oils, substituted aromatic organic liquids such as dioctyl phthalate; kerosene; dialkyl amides of various fatty acids, particularly the dimethyl amides; and glycol ethers such as the n-butyl ether, ethyl ether or methyl ether of diethylene glycol, and the methyl ether of triethylene glycol and the like. Mixtures of two or more organic liquids may also be employed in the preparation of the emulsifiable concentrate. Surface-active emulsifying agents are typically employed in liquid formulations and in an amount of from 0.1 to 20 percent by weight based on the combined weight of the emulsifying agents.

Aqueous suspensions comprise suspensions of one or more water-insoluble active ingredients dispersed in an aqueous vehicle at a concentration in the range from about 5 to about 50 weight percent, e.g., about 0.1 weight percent, and intermediate ranges, based on the total weight of the aqueous suspension. Suspensions are prepared by finely grinding one or more of the active ingredients, and vigorously mixing the ground material into a vehicle comprised of water and surfactants chosen from the same types discussed above. Other components, such as inorganic salts and synthetic or natural gums, may also be added to increase the density and viscosity of the aqueous vehicle. It is often most effective to grind and mix at the same time by preparing the aqueous mixture and homogenizing it in an implement such as a sand mill, ball mill, or piston-type homogenizer.

Aqueous emulsions comprise emulsions of one or more water-insoluble active ingredients emulsified in an aqueous vehicle at a concentration typically in the range from about 5 to about 50 weight percent, based on the total weight of the aqueous emulsion. If the active ingredient is a solid it must be dissolved in a suitable water-immiscible solvent prior to the preparation of the aqueous emulsion. Emulsions are prepared by emulsifying the liquid active ingredient or water-immiscible solution thereof into an aqueous medium typically with inclusion of surfactants that aid in the formation and stabilization of the emulsion as described above. This is often accomplished with the aid of vigorous mixing provided by high shear mixers or homogenizers.

Granular formulations usually contain from about 0.5 to about 10 weight percent, based on the total weight of the granular formulation of the active ingredient(s), dispersed in an inert carrier which consists entirely or in large part of coarsely divided inert material such as attapulgite, bentonite, diatomite, clay or a similar inexpensive substance. Such formulations are usually prepared by dissolving the active ingredients in a suitable solvent and applying it to a granular carrier which has been preformed to the appropriate particle size, in the range of from about 0.5 to about 3 mm. A suitable solvent is a solvent in which the compound is substantially or completely soluble. Such formulations may also be prepared by making a dough or paste of the carrier and the compound and solvent, and crushing and drying to obtain the desired granular particle.

Dusts can be prepared by intimately mixing one or more of the active ingredients in powdered form with a suitable dusty agricultural carrier, such as, for example, kaolin clay, ground volcanic rock, and the like. Dusts can suitably contain from about 1 to about 10 weight percent of the compounds, based on the total weight of the dust.

The formulations may additionally contain adjuvant surfactants to enhance deposition, wetting and penetration of the active ingredients onto the target site such as a crop or organism. These adjuvant surfactants may optionally be employed as a component of the formulation or as a tank mix. The amount of adjuvant surfactant will typically vary from 0.01 to 1.0 percent by volume, based on a spray-volume of water, preferably 0.05 to 0.5 volume percent. Suitable adjuvant surfactants include, but are not limited to ethoxylated nonyl phenols, ethoxylated synthetic or natural alcohols, salts of the esters of sulfosuccinic acids, ethoxylated organosilicones, ethoxylated fatty amines and blends of surfactants with mineral or vegetable oils.

The formulations may optionally include combinations that contain one or more fungicides or pesticidal compounds. Pesticidal compounds may be insecticides, nematicides, miticides, arthropodicides, bactericides or combinations thereof that are compatible or synergistic with the compounds of the presently disclosed subject matter in the medium selected for application, and not antagonistic to the activity of the tritriacontene or toxic to honey bees. The tritriacontene of the presently disclosed subject matter and the pesticidal compound or fungicide in the combination can generally be present in a weight ratio of from 1:100 to 100:1.

As used herein, in relation to compositions comprising a tritriacontene, the term “an effective amount for inducing hygienic behavior in honey bees,” refers to the amount of the tritriacontene necessary to induce hygienic behavior in honey bees. As used herein, in relation to compositions comprising mite-infested brood extract, the term “an effective amount for inducing hygienic behavior in honey bees,” refers to the amount of the mite-infested brood extract necessary to induce hygienic behavior in honey bees.

The compositions of the presently disclosed subject matter herein may have broad ranges of uses to treat a hive or control against pests, diseases, pathogens, or infestations harmful to honey bees. The compositions of the presently disclosed subject matter herein may have broad ranges of uses as a prophylactic treatment of a hive; particularly, wherein the hygienic behavior results in inspection of the hive. In one embodiment, the prophylactic treatment is to reduce the likelihood of colony collapse disorder or severe Varroa mite infestation. In another embodiment, the hygienic behavior comprises eating diseased brood or diseased honey bees, removing diseased brood or diseased honey bees from hive cells, removing pests or parasites, uncapping hive cells, or uncapping and recapping hive cells. In a further embodiment, the hygienic behavior results in survival of a honey bee colony. In another embodiment, the hygienic behavior results in suppression of mite reproduction, decreased mite survival, or suppression of a mite infestation.

The exact amount of the active material to be applied can be dependent on several factors including, but not limited to, the specific active material being applied; the particular action desired; the pathogen, disease, or pest to be controlled or treated against; the stage of growth thereof; the number of honey bees to be induced; the particular honey bee species; the particular honey bee breed; thresholds to avoid toxicity to the brood or hive generally; the type of formulation employed; and the method of application including, for example, dilution and rate of application and equipment employed; climate conditions. The effective amount of the tritriacontene can readily be determined by those skilled in the art. This amount will generally be from about 0.1 to about 1000 parts per million (ppm), with 1 to 500 ppm being preferred. In another embodiment, the effective amount of the tritriacontene is within the range of 1 nanogram to 1 gram per hive cell; more particularly, within the range a single tritriacontene molecule to 1 gram per hive cell. The effective amount of the mite-infested brood extract can readily be determined by those skilled in the art. This amount will generally be from about 10,000 to about 1×10⁻⁷ parts per million (ppm), with 1×10⁻² to 1×10⁻⁴ ppm being preferred.

Generally, when the compositions disclosed in this document are used in a formulation, such formulation can also contain other components. These components include, but are not limited to, (this is a non-exhaustive and non-mutually exclusive list) wetters, spreaders, stickers, penetrants, buffers, sequestering agents, drift reduction agents, compatibility agents, anti-foam agents, cleaning agents, rheology agents, stabilizers, dispersing agents and emulsifiers. A few components are described forthwith.

A wetting agent is a substance that when added to a liquid increases the spreading or penetration power of the liquid by reducing the interfacial tension between the liquid and the surface on which it is spreading. Wetting agents are used for two main functions in agrochemical formulations: during processing and manufacture to increase the rate of wetting of powders in water to make concentrates for soluble liquids or suspension concentrates; and during mixing of a product with water in a spray tank to reduce the wetting time of wettable powders and to improve the penetration of water into water-dispersible granules. Examples of wetting agents used in wettable powder, suspension concentrate, and water-dispersible granule formulations are: sodium lauryl sulphate; sodium dioctyl sulphosuccinate; alkyl phenol ethoxylates; and aliphatic alcohol ethoxylates.

A dispersing agent is a substance which adsorbs onto the surface of particles and helps to preserve the state of dispersion of the particles and prevents them from reaggregating. Dispersing agents are added to agrochemical formulations to facilitate dispersion and suspension during manufacture, and to ensure the particles redisperse into water in a spray tank. They are widely used in wettable powders, suspension concentrates and water-dispersible granules. Surfactants that are used as dispersing agents have the ability to adsorb strongly onto a particle surface and provide a charged or steric barrier to reaggregation of particles. The most commonly used surfactants are anionic, non-ionic, or mixtures of the two types. For wettable powder formulations, the most common dispersing agents are sodium lignosulphonates. For suspension concentrates, very good adsorption and stabilization are obtained using polyelectrolytes, such as sodium naphthalene sulphonate formaldehyde condensates. Tristyrylphenol ethoxylate phosphate esters are also used. Non-ionics such as alkylarylethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersing agents for suspension concentrates. In recent years, new types of very high molecular weight polymeric surfactants have been developed as dispersing agents. These have very long hydrophobic ‘backbones’ and a large number of ethylene oxide chains forming the ‘teeth’ of a ‘comb’ surfactant. These high molecular weight polymers can give very good long-term stability to suspension concentrates because the hydrophobic backbones have many anchoring points onto the particle surfaces. Examples of dispersing agents used in agrochemical formulations are: sodium lignosulphonates; sodium naphthalene sulphonate formaldehyde condensates; tristyrylphenol ethoxylate phosphate esters; aliphatic alcohol ethoxylates; alky ethoxylates; EO-PO block copolymers; and graft copolymers.

An emulsifying agent is a substance which stabilizes a suspension of droplets of one liquid phase in another liquid phase. Without the emulsifying agent the two liquids would separate into two immiscible liquid phases. The most commonly used emulsifier blends contain alkylphenol or aliphatic alcohol with 12 or more ethylene oxide units and the oil-soluble calcium salt of dodecylbenzene sulphonic acid. A range of hydrophile-lipophile balance (“HLB”) values from 8 to 18 will normally provide good stable emulsions. Emulsion stability can sometimes be improved by the addition of a small amount of an EO-PO block copolymer surfactant.

A solubilizing agent is a surfactant which will form micelles in water at concentrations above the critical micelle concentration. The micelles are then able to dissolve or solubilize water-insoluble materials inside the hydrophobic part of the micelle. The types of surfactants usually used for solubilization are non-ionics: sorbitan monooleates; sorbitan monooleate ethoxylates; and methyl oleate esters.

Surfactants are sometimes used, either alone or with other additives such as mineral or vegetable oils as adjuvants to spray-tank mixes to improve the biological performance of the composition on the target. The types of surfactants used for bioenhancement depend generally on the nature and mode of action of the composition. However, they are often non-ionics such as: alkyl ethoxylates; linear aliphatic alcohol ethoxylates; aliphatic amine ethoxylates.

Organic solvents are used mainly in the formulation of emulsifiable concentrates, ULV formulations, and to a lesser extent granular formulations. Sometimes mixtures of solvents are used. The first main groups of solvents are aliphatic paraffinic oils such as kerosene or refined paraffins. The second main group and the most common comprises the aromatic solvents such as xylene and higher molecular weight fractions of C9 and C10 aromatic solvents. Chlorinated hydrocarbons are useful as cosolvents to prevent crystallization of the compositions when the formulation is emulsified into water. Alcohols are sometimes used as cosolvents to increase solvent power.

Thickeners or gelling agents are used mainly in the formulation of suspension concentrates, emulsions and suspoemulsions to modify the rheology or flow properties of the liquid and to prevent separation and settling of the dispersed particles or droplets. Thickening, gelling, and anti-settling agents generally fall into two categories, namely water-insoluble particulates and water-soluble polymers. It is possible to produce suspension concentrate formulations using clays and silicas. Examples of these types of materials, include, but are limited to, montmorillonite, e.g. bentonite; magnesium aluminum silicate; and attapulgite. Water-soluble polysaccharides have been used as thickening-gelling agents for many years. The types of polysaccharides most commonly used are natural extracts of seeds and seaweeds are synthetic derivatives of cellulose. Examples of these types of materials include, but are not limited to, guar gum, locust bean gum; carrageenam; alginates; methyl cellulose; sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC). Other types of anti-settling agents are based on modified starches, polyacrylates, polyvinyl alcohol and polyethylene oxide. Another good anti-settling agent is xanthan gum.

Microorganisms cause spoilage of formulated products. Therefore preservation agents are used to eliminate or reduce their effect. Examples of such agents include, but are not limited to: propionic acid and its sodium salt; sorbic acid and its sodium or potassium salts; benzoic acid and its sodium salt; p-hydroxy benzoic acid sodium salt; methyl p-hydroxy benzoate; and 1,2-benzisothiazalin-3-one (BIT).

The presence of surfactants, which lower interfacial tension, often causes water-based formulations to foam during mixing operations in production and in application through a spray tank. In order to reduce the tendency to foam, anti-foam agents are often added either during the production stage or before filling into bottles. Generally, there are two types of anti-foam agents, namely silicones and non-silicones. Silicones are usually aqueous emulsions of dimethyl polysiloxane while the non-silicone anti-foam agents are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the anti-foam agent is to displace the surfactant from the air-water interface.

“Green” agents (e.g., adjuvants, surfactants, solvents) can reduce the overall environmental footprint of crop protection formulations. Green agents are biodegradable and generally derived from natural and/or sustainable sources, e.g. plant and animal sources. Specific examples are: vegetable oils, seed oils, and esters thereof, also alkoxylated alkyl polyglucosides.

For further information see “CHEMISTRY AND TECHNOLOGY OF AGROCHEMICAL FORMULATIONS” edited by D. A. Knowles, copyright 1998 by Kluwer Academic Publishers.

II. METHODS FOR INDUCING HYGIENIC BEHAVIOR

In other embodiments, the presently disclosed subject matter is directed to a method of inducing hygienic behavior in honey bees, the method comprising contacting hive cells with a composition comprising a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees. In other embodiments, the presently disclosed subject matter is directed to a method of inducing hygienic behavior in honey bees, the method comprising contacting hive cells with a composition comprising a mite-infested brood extract and an agriculturally acceptable diluent or carrier, wherein the mite-infested brood extract is in an effective amount for inducing hygienic behavior in honey bees. In further embodiments, the composition, the tritriacontene, the mite-infested brood extract, the agriculturally acceptable diluent or carrier, the effective amount for inducing hygienic behavior in honey bees, and other terms may be as described herein. The methods of the presently disclosed subject matter may have broad ranges of uses to treat a hive or control against pests, diseases, pathogens, or infestations harmful to honey bees. In another embodiment, the hygienic behavior results in suppression of mite reproduction, decreased mite survival, or suppression of a mite infestation. In another embodiment, the hygienic behavior results in survival of a honey bee colony. In another embodiment, the hive cells are capped hive cells or uncapped hive cells. In another embodiment, the hive cells are worker-brood cells, drone-brood cells, or queen bee cells. In a further embodiment, the honey bees are of the species Apis mellifera or Apis cerana.

As social insects, honey bees complement individual immunity with mechanisms of social immunity for defense against pathogens and parasites. Honey bees are able to reduce parasite and pathogen loads through age-specific sanitary activities such as hygienic behavior. The complete mechanism for hygienic behavior is not completely understood, but can be measured, for instance, by frequency of certain behaviors in honey bees, such as eating diseased brood or diseased honey bees, removing diseased brood or diseased honey bees from hive cells, removing pests or parasites, uncapping hive cells, or uncapping and recapping hive cells. See for example. [8, 9, 11] Hygienic behavior has been described by some as the detection, uncapping and/or removal of diseased brood from the hive. [182] As used herein, the term “hygienic behavior” includes those described herein and known in the art, for example, without limiting the foregoing, as described in Spivak, M., (1996) Honey bee hygienic behavior and defense against Varroa jacobsoni. Apidologie 27: 245-260.

In one embodiment, the hygienic behavior comprises eating diseased brood or diseased honey bees, removing diseased brood or diseased honey bees from hive cells, removing pests or parasites, uncapping hive cells, or uncapping and recapping hive cells. In another embodiment, the hygienic behavior comprises uncapping a hive cell, removing pests or parasites, and recapping the hive cell. In a further embodiment, the diseased brood or diseased honey bees are infested with a pest or parasite; infected with a pathogen; or damaged. In yet another embodiment the diseased brood are eggs, larvae, or pupae.

In a further embodiment, the pests or parasites are mites, wax moth, small hive beetle, or Nosema. In a further embodiment, the mites are mites of the genus Varroa; particularly wherein the mites are mites of the species Varroa destructor or Varroa jacobsoni. In a further embodiment, the mites are mites of the genus Acarapis: particularly wherein the mites are mites of the species Acarapis woodi (also known as tracheal mite). In another embodiment, the mites are mites of the genus Tropilaelaps.

Varroa destructor is an obligate, ectoparasitic honey bee mite, arguably the most important threat to honey bee health and apiculture today. [31-32] During their reproductive stage, female foundress mites enter honey brood cells just before capping and bury themselves in the brood food at the base of the cell. After about six hours when the food has been consumed by the bee brood, the mite emerges and establishes a feeding site on the brood, from which it sucks hemolymph. [198] Approximately 70 hours after cell capping the foundress mite begins to lay eggs, the first of which is haploid and develops into a male. Diploid eggs are then laid at approximately 30-hour intervals. These develop into females which mate with the waiting male such that by the end of honey bee development up to four (worker cell) or five (drone cell) fertilized female Varroa may emerge with the emerging honey bee to repeat the cycle. [31] Varroa act as a physical burden to the bee, reducing body weight and protein levels primarily through the sucking of hemolymph. [44, 199, 200, 201, 202]

While the physical burden of Varroa is problematic to honey bee health, it is merely one of many honey bee threats associated with Varroa. Varroa transmit diseases to honey bees [60, 135, 159, 161] and have been associated with both viral amplification and honey bee disease susceptibility. [51, 60] Varroa mites enter honey bee colonies as adults, presumably with returning foragers or drifting workers from other colonies. Inside the nest, the mature, fertilized Varroa females enter a brood cell that contains a worker or drone larvae that is about to be closed with a wax cap. After the cell is sealed, the mite emerges from hiding and establishes a feeding site that it will share with its offspring. [35] While the host is undergoing its final molt from the 5^(th) larval instar to pupae, the Varroa foundress initiates egg-laying, first producing an unfertilized male egg and then subsequently fertilized female eggs every 30 hours. These offspring develop and sib-mate before leaving the cell when the adult honey bee opens the wax capping to emerge. [36, 37]

Varroa mites only reproduce on drone brood in A. cerana honey bee hosts but are able to utilize worker and drone brood to complete their life cycle in A. mellifera honey bees. This difference in host utilization is presumably due to longer development and less hygienic behavior of A. mellifera honey bees. [38, 39] Varroa directly impairs colony function in A. mellifera honey bees, while only curtailing the drone production of A. cerana honey bees. Moreover, the persistent worker brood production allows for the build-up of higher Varroa population densities in A. mellifera colonies. [40]

Varroatosis is caused by the mite parasitizing honey bees, e.g. drones and queen bees, larvae and pupae. Although Varroa causes physical and physiological damage when feeding on the honey bee hemolymph [44], its most serious impact on honey bee health is caused by enabling viral diseases. [45] Varroatosis produces great damage in apiculture due to the acute debilitation and high mortality of the members of the honey bee colony. Also, Varroa is an effective vector of viruses [30, 31] and possibly other microorgansms. [43] V. destructor increases the virulence of viruses [46] and may lead to fatal outbreaks. [47, 48] Specifically, more virulent, strains of viruses are selectively favored. [49] Varroa feeding may also activate latent viruses. [31, 50]

Small hive beetle reproduces in a hive and is a damaging pest of beeswax combs, comb honey and bee-collected pollen. The females will lay egg masses in protected crevasses in the hive. The larva feed on the honey and pollen. If the infestation is severe enough the bees will abandon the hive. As the beetles move about the hive they defecate forming a slimy mess that results in the honey fermenting.

Wax moth larvae are very destructive and can quickly destroy stored beeswax combs. They tunnel and chew through combs, particularly combs that have contained brood and pollen. Developing honey bee pupae are exposed when wax moth larvae partly remove the cell caps, a condition known as bald brood. Worker bees chew the remainder of the capping thereby fully exposing the heads of the pupae that continue to develop normally. The lines of bald brood follow the direction of the wax moth's travel. Some honey bee pupae nearing maturity may have deformed legs or wings. One of the causes of this deformity is a result of wax moth excreta affecting the final molt of the pupa before its emergence from the cell.

Nosema is a parasitic protozoa, caused by the microsporidian Nosema apis or Nosema ceranae that resides in the gut of the bee. The parasites damage the hosts by destroying internal organs. Inside the cell of the bee's gut, Nosema reproduces by forming spores, which are passed within the bee's waste. Bees will begin to expel waste in the hive and on the outside; and can cause rapid colony decline.

In another embodiment, the pathogen is a bacterium, fungus, or virus. In one embodiment the pathogen is Ascosphaera apis; particularly wherein Ascosphaera apis causes chalkbrood. In one embodiment, the pathogen is Aspergillus fumigatus, Aspergillus flavus, or Aspergillus niger; more particularly wherein Aspergillus fumigatus, Aspergillus flavus, or Aspergillus niger causes stonebrood. In one embodiment, the bacterium is Paenibacillus larvae (formerly classified as Bacillus larvae and Paenibacillus larvae ssp larvae/pulvifaciens); more particularly wherein Paenibacillus larvae causes American foulbrood. In one embodiment, the bacterium is Melissococcus plutonius; more particularly wherein Melissococcus plutonius causes European foulbrood. In one embodiment, the virus is of the family of cripaviridae viruses; more particularly, wherein the virus is chronic paralysis virus. In one embodiment, the virus is of the family of dicistroviridae viruses; more particularly, wherein the virus is acute bee paralysis virus, Israeli acute paralysis virus, Kashmir bee virus, or Black queen cell virus. In one embodiment, the virus is Cloudy wing virus. Sacbrood virus, or Perina nuda. In another embodiment, the virus is Morator aetatulas; more particularly, wherein Morator aetatulas causes sacbrood disease. In one embodiment, the virus is of the family Iflaviridae; more particularly, wherein the virus is Deformed wing virus. In one embodiment, the virus is of the family Iridoviridae; more particularly, wherein the virus is invertebrate iridescent virus type 6 (IIV-6). In one embodiment, the virus is of the family Secoviridae; more particularly, wherein the virus is tobacco ringspot virus. In one embodiment, the virus is slow paralysis virus.

Deformed wing virus (DWV), Kashmir bee virus (KBV), sacbrood virus (SBV), acute bee paralysis virus (ABPV), slow paralysis virus (SPV), and Israeli acute paralysis virus (IAPV) have been associated with Varroa [42, 57, 58] Many of these viruses have presumably alternative modes of transmission 1591 but the effective horizontal transmission by the Varroa vector has profound implications for the viruses that are found in the honey bee hosts. [60]

Chalkbrood is a disease caused by the fungus Ascosphaera apis that turns the body of an infected bee larva into fungal cells which eventually produce millions of spores. Infected larvae become overgrown with a white cotton-like mycelium and eventually dry to a hard, white or gray shrunken mass (thus the name Chalkbrood) referred to as a mummy. The fruit-bodies of the fungus develop on the gray-colored mummies, and the spores released from the spore capsules can enter the air of the beehive. The disease is spread through local populations by adult bees emerging from contaminated media. It has been shown that a single adult bee may carry from 50 to 300 million spores on its body surface after having chewed through a single diseased cadaver as it extracts itself from the cell or nesting material. Once the disease becomes established in an area it increases rapidly because of the reuse of contaminated nesting media in successive years. As a result of infection, the colonies fail to grow to a sufficiently large size, their resistance becomes impaired and their honey-producing capacity decreases to a degree depending on the severity of the mycotic infection. Over the past several years various control measures have been developed but none have been completely effective or economically practical. Sterilization of nesting media has been attempted by use of dry chlorine or bleach, convection heat, and microwave exposure. Also, surface sterilization of adult bees consisting of a bee bath in sodium hypochlorite or iodine has been employed. Dusting with general antibiotics and fungicides in such a manner that they are ingested by the adult bee also has met with little success. A related infection, Stonebrood disease (forming stone-hard larvae) is caused by the fungus Aspergillus flavus and related species.

American foulbrood is caused by the bacterium Paenibacullus larvae, which can remain viable indefinitely on beekeeping equipment. It infects the gut of worker, drone and queen larvae and, while it may not destroy a colony in the first year, if left unchecked may ultimately lead to the death of the colony. The main method of treatment is with the antibiotic oxytetracycline, administered in various forms with a sugar carrier. However, there are many problems associated with administration of oxytetracycline, including problems related to stability, antibiotic contamination of the honey, the possibility of killing open brood on the face of brood combs, and unevenness of dosing.

European Foulbrood disease is caused by the bacterium Melissococcus pluton, which is fed to the worker, drone and queen larvae by nurse bees. Diseased colonies fail to increase normally so that no surplus honey, in excess of that needed by the colony to survive, is available for the beekeeper. Oxytetracycline is also used for treating such diseased colonies.

“Damaged” refers to physically damaged, physiologically damaged, health-compromised, and/or immune system-compromised, or dead. The foregoing types of damage can be the result of, without limiting the foregoing, a parasite, pest, pathogen, environmental change-related stresses, malnutrition, or exposure to contaminated food (e.g. for example to pesticide laden pollen), pesticides (e.g. neonicotinoids such as imidacloprid, clothianidin, and thiamethoxam; neonicotinamides; carbamates), insecticides (e.g. organophosphates), fungicides, miticides (e.g. acaricides, such as coumaphos, tau-fluvalinate, flumethrin, and amitraz), or other toxic chemicals.

For example, without limiting the foregoing, honey bees with Varroatosis or dysentery are damaged. For example, without limiting the foregoing, chilled brood or bald brood are damaged. Removal rates that correspond to the level of brood health support the existence of the evolution of a damage-dependent hygienic response. [93, 156] Damage-dependent hygiene is also supported by evidence that immune response affects honey bee cuticular chemicals. [112] Existence of a damage-dependent response to Varroa has recently been supported by evidence of mite-virulence dependent hygienic removal. [179]

Miticides used to control Varroa infestations, such as fluvalinate and coumaphos, have been found to have lethal and sublethal effects on honey bee queens, workers, and drones. [2, 66, 132, 154, 162, 177, 181] For example, moderate doses of fluvalinate in the hive have been associated with reduced queen weight [68] and reduced drone weight and number of spermatozoa. [203] Even low doses of coumaphos have been associated with increased queen mortality, physical deformities, reduced body and ovary weight, and atypical behavior [68, 133, 177] and moderate coumaphos exposure has been linked to reduction of drone sperm vitality. [204] Synergistic effects of fluvalinate and coumaphos have been measured, where the toxicity of each chemical is significantly increased in bees previously exposed to the other. [172] The lipophilic nature of both the synthetic pyrethroid fluvalinate and the organophosphate coumaphos leads to high absorption and accumulation of the chemicals in hives, especially in wax, meaning that exposure of bees to these and similar compounds increases with time and number of chemical treatments. A 2007 study of residues in honey bee hives found 46 pesticides in 108 pollen samples, and 20 pesticides in 88 wax samples, with over 55% of pollen and 100% of wax samples containing the most concentrated pesticides: the miticides fluvalinate and coumaphos. [62] Furthermore, immunosuppression caused by chemical exposure makes honey bees more susceptible to parasites like Varroa, as well as to the pathogens they vector. [64, 70, 181, 184]

In another embodiment, the hygienic behavior comprises uncapping hive cells of healthy brood or diseased brood; more particularly, wherein the hygienic behavior further comprises recapping the hive cells of healthy brood; particularly, wherein the hygienic behavior further comprises removing diseased brood or pests or parasites; particularly, wherein the hygienic behavior further comprises removing diseased brood or pests or parasites, and recapping the hive cells. The methods of the presently disclosed subject matter herein may have broad ranges of uses as a prophylactic treatment of a hive; particularly, wherein the hygienic behavior results in inspection of the hive. For example, without limiting the foregoing, nurse bees will remove Varroa-infested brood but simply recap healthy brood. [96] In one embodiment, the prophylactic treatment is to reduce the likelihood of colony collapse disorder or severe Varroa mite infestation.

In another embodiment, contacting hive cells with a composition comprises spraying, dusting, dipping, spotting, or fumigating. In another embodiment, the contacting of the hive cells is on one or more days after the hive cells are capped. In another embodiment, the contacting of the hive cells is on one or more days before the hive cells are capped. The hive cells may be artificial or natural compartments. The hive cells may be part of combs that are natural or artificial (including combs made of wax, resin, plastic, metal, wax-coated plastic). The hive cells are not necessarily part of a hive.

III. METHODS FOR SELECTING BEES EXHIBITING HYGIENIC BEHAVIOR

Current methods for selectively breeding bees for hygienic behavior result in bees that have varying degrees of sensitivity to diseased broods. Reasons for lack of efficacy include non mite-specificity of selection processes. [92, 95, 183] For example, the olfactory trigger for hygienic removal of mite-infested and other live brood may be significantly lower than that of dead brood. [156, 100, 183] As a result, the threshold for olfactory response needs to be lower for mite-removal than it does for removal of freeze-killed brood or brood infected with more virulent honey bee diseases such as Chalkbrood or American Foulbrood (caused by the fungus Ascosphaera apis and the bacterium Paenibacullus larvae, respectively). Also, existence of the evolution of a damage-dependent hygienic response is supported by removal rates that correspond to the level of brood health. [156, 93]

The presently disclosed subject matter provides methods for selecting bees exhibiting hygienic behavior as described more fully below and relates to findings that a tritriacontene compound triggers hygienic behavior in honey bees, and mite-infested brood extract triggers hygienic behavior in honey bees.

In additional embodiments, the presently disclosed subject matter is directed to a method for selecting one or more honey bee(s) exhibiting hygienic behavior comprising a) applying a tritriacontene composition to a set of hive cells; b) performing an assay to identify a hygienic colony, wherein the assay comprises exposing the set of hive cells to a test colony; and c) selecting one or more honey bee(s) from an identified hygienic colony, wherein the selected one or more honey bee(s) exhibit hygienic behavior.

In additional embodiments, the presently disclosed subject matter is directed to a method for selecting one or more honey bee(s) exhibiting hygienic behavior comprising a) applying a mite-infested brood extract composition to a set of hive cells; b) performing an assay to identify a hygienic colony, wherein the assay comprises exposing the set of hive cells to a test colony; and c) selecting one or more honey bee(s) from an identified hygienic colony, wherein the selected one or more honey bee(s) exhibit hygienic behavior.

In further embodiments, the set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the identified hygienic colony comprises honey bees exhibiting hygienic behavior; more particularly wherein the hygienic behavior comprises eating diseased brood or diseased honey bees, removing diseased brood or diseased honey bees from hive cells, removing pests or parasites, uncapping hive cells, or uncapping and recapping hive cells.

In one embodiment, each of the selected honey bee(s) is a queen bee or drone bee. In further embodiments, the tritriacontene composition, the mite-infested brood extract, the diseased brood or diseased honey bees, pests or parasites, and other terms may be as described herein. In another embodiment, applying the tritriacontene composition or the mite-infested brood extract comprises spraying, dusting, dipping, spotting, or fumigating. In another embodiment, the hive cells are worker-brood cells, drone-brood cells, or queen bee cells.

In another embodiment, the assay further comprises i) determining the amount of emptied hive cells in the set of hive cells; and ii) identifying a hygienic colony, wherein a test colony is a hygienic colony if at least 85% of the set of hive cells are emptied; more particularly, wherein a test colony is a hygienic colony if at least 90% of the set of hive cells are emptied; more particularly, wherein a test colony is a hygienic colony if at least 95% of the set of hive cells are emptied. In another embodiment, the emptied hive cells have no eggs, larvae, or pupae, or contain partially eaten larvae or pupae. In another embodiment, the emptied hive cells have no diseased honey bees, or contain partially eaten diseased honey bees. In another embodiment, the emptied hive cells have no pests or parasites, or contain eaten pests or parasites. In another embodiment, the emptied hive cells are capped hive cells or uncapped hive cells.

In another embodiment, the hive cells are capped hive cells and the assay further comprises i) determining the amount of uncapped and/or recapped hive cells in the set of capped hive cells; and ii) identifying a hygienic colony, wherein a test colony is a hygienic colony if at least 85% of the set of capped hive cells are uncapped and/or recapped; more particularly, wherein a test colony is a hygienic colony if at least 90% of the set of capped hive cells are uncapped and/or recapped; more particularly, wherein a test colony is a hygienic colony if at least 95% of the set of capped hive cells are uncapped and/or recapped. In a further embodiment, the capped hive cells are empty.

In another embodiment, the method further comprises d) mating a selected honey bee with one or more honey bee(s) from at least one separately identified hygienic colony to produce offspring; more particularly wherein at least one separately identified hygienic colony was bred according to the same method; or more particularly wherein at least one separately identified hygienic colony was bred or identified by a method based on freeze-killed brood, suppression of mite reproduction, or removal of damaged brood. In another embodiment, a queen bee is selected and mated: (a) naturally with one or more drones from at least one separately identified hygienic colony; or (b) artificially inseminated with semen from one or more drones from at least one separately identified hygienic colony.

The bees may be mated in a manner known in the art; for example, without limiting the foregoing, by means of conventional breeding processes (e.g. open mating in drone congregation areas of breeds of specifically enriched traits) and mating equipment (e.g. artificial insemination devices).

In another embodiment, the method further comprises e) raising the offspring, f) applying a tritriacontene composition to a second set of hive cells, g) performing a second assay to identify whether the raised offspring is a hygienic colony, wherein the second assay comprises exposing the second set of hive cells to the raised offspring, and h) selecting one or more honey bee(s) from an identified hygienic colony, wherein the selected one or more honey bee(s) exhibit hygienic behavior.

In another embodiment, the method further comprises e) raising the offspring, f) applying a mite-infested brood extract composition to a second set of hive cells, g) performing a second assay to identify whether the raised offspring is a hygienic colony, wherein the second assay comprises exposing the second set of hive cells to the raised offspring, and h) selecting one or more honey bee(s) from an identified hygienic colony, wherein the selected one or more honey bee(s) exhibit hygienic behavior.

The offspring may be raised in a manner known in the art; for example, without limiting the foregoing, by means of conventional processes of queen rearing and drone rearing to propagate the trait.

In further embodiments, the second set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the identified hygienic colony comprises honey bees exhibiting hygienic behavior; more particularly wherein the hygienic behavior comprises eating diseased brood or diseased honey bees, removing diseased brood or diseased honey bees from hive cells, removing pests or parasites, uncapping hive cells, or uncapping and recapping hive cells. In further embodiments, the pests or parasites, the diseased brood, the diseased honey bees, and other terms may be as described herein.

In another embodiment, the second assay further comprises i) determining the amount of emptied hive cells in the second set of hive cells; and ii) identifying a hygienic colony, wherein the raised offspring is a hygienic colony if at least 85% of the second set of hive cells are emptied; more particularly, wherein the raised offspring is a hygienic colony if at least 90% of the second set of hive cells are emptied; more particularly, wherein the raised offspring is a hygienic colony if at least 95% of the second set of hive cells are emptied. In another embodiment, the emptied hive cells have no eggs, larvae, or pupae, or contain partially eaten larvae or pupae. In another embodiment, the emptied hive cells have no diseased honey bees, or contain partially eaten diseased honey bees. In another embodiment, the emptied hive cells have no pests or parasites, or contain eaten pests or parasites. In another embodiment, the emptied hive cells are capped hive cells or uncapped hive cells.

In another embodiment, the hive cells are capped hive cells and the second assay further comprises i) determining the amount of uncapped and/or recapped hive cells in the second set of capped hive cells; and ii) identifying a hygienic colony, wherein the raised offspring is a hygienic colony if at least 85% of the second set of capped hive cells are uncapped and/or recapped; more particularly, wherein the raised offspring is a hygienic colony if at least 90% of the second set of capped hive cells are uncapped and/or recapped; more particularly, wherein the raised offspring is a hygienic colony if at least 95% of the second set of capped hive cells are uncapped and/or recapped. In a further embodiment, the capped hive cells are empty.

The assay and the second assay each independently may comprise steps known in the art for selective breeding of hygienic behavior, including those for augmenting hive resistance to Varroa. [75, 87, 92, 155, 183] For example, HYG breeds are selected for based on the removal of freeze-killed brood. In some embodiments, the assay and the second assay each independently may further comprise i) freeze-killing a portion of a brood, ii) determining the amount of hive cells uncapped and containing no diseased brood; and iii) identifying a hygienic colony, wherein a test colony is a hygienic colony if at least 85% of the set of hive cells are uncapped and containing no diseased brood; more particularly, wherein a test colony is a hygienic colony if at least 90% of the set of hive cells are uncapped and containing no diseased brood; more particularly, wherein a test colony is a hygienic colony if at least 95% of the set of hive cells are uncapped and containing no diseased brood. In another embodiment, VSH breeds are selected for based on apparent suppression of mite reproduction. In some embodiments, the set of hive cells contains diseased brood infested with mites of the species Varroa destructor, and the assay and the second assay each independently may further comprises i) determining the amount of hive cells containing no diseased brood; and ii) identifying a hygienic colony, wherein a test colony is a hygienic colony if at least 85% of the set of hive cells contain no diseased brood; more particularly, wherein, a test colony is a hygienic colony if at least 90% of the set of hive cells contain no diseased brood; more particularly, wherein, a test colony is a hygienic colony if at least 95% of the set of hive cells contain no diseased brood.

In some embodiments, the identified hygienic colony as presently disclosed in the subject matter herein may have broad ranges of uses to treat a hive or control against pests, diseases, pathogens, or infestations harmful to honey bees. In some embodiments, the identified hygienic colony as presently disclosed in the subject matter herein may have broad ranges of uses as a prophylactic treatment of a hive; particularly, wherein the hygienic behavior results in inspection of the hive. In one embodiment, the prophylactic treatment is to reduce the likelihood of colony collapse disorder or severe Varroa mite infestation.

In some embodiments, the identified hygienic colony as presently disclosed in the subject matter herein may have broad ranges of uses to produce, for example, honey, nectar, beeswax, pollen, or propolis. In some embodiments, the identified hygienic colony as presently disclosed in the subject matter herein may have broad ranges of uses to pollinate plants and crops, for example without limiting the foregoing, insect-pollinated plants and crops; more particularly fruits, vegetables, or nuts. Non-limiting examples of plants and crops include acerola, adzuki bean, allspice, almond, almonds, apricot, apple, avocado, azarole, beet, black currant, blackberry, blueberry, boysenberry, broccoli, Brussels sprouts, buckwheat, cabbage, cantaloupe, caraway, cardamom, carrot, cashew, cauliflower, celery, chestnut, citrus tree, clover, coconut, coffee, coriander, cotton, crownvetch, cucumber, elderberry, feijoa, fennel, flax, grape, green bean, guar bean, guava, haricot bean, hyacinth bean, jujube, kidney bean, kiwifruit, lemon, lima bean, lime, longan, loquat, lupine, lychee, macadamia, mango, melon, mungo bean, mustard, nectarine, okra, onion, papaya, peach, pear, pear, peas, peppers, persimmon, plum, pomegranate, quince, rambutan, rapeseed, raspberry, red currant, rose hips, rowanberry, safflower, sainfoin, scarlet runner bean, service tree, sesame, sour cherry, squash, starfruit, strawberry tree, strawberry, string bean, sunflower, sweet cherry, tamarind, tangelo, tomato, turnip, or watermelon.

IV. ASSAYS

In some embodiments, the presently disclosed subject matter is directed to a method for assessing the degree of hygienic behavior within a honey bee colony comprising a) applying a tritriacontene composition to a set of hive cells; b) exposing the set of hive cells to a honey bee colony; and c) determining the amount of emptied hive cells in the set of hive cells; wherein a higher amount of the set of hive cells that are emptied is associated with a greater degree of hygienic behavior. In further embodiments, the set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the emptied hive cells are capped hive cells or uncapped hive cells.

In some embodiments, the presently disclosed subject matter is directed to a method for assessing the degree of hygienic behavior within a honey bee colony comprising a) applying a mite-infested brood extract composition to a set of hive cells; b) exposing the set of hive cells to a honey bee colony; and c) determining the amount of emptied hive cells in the set of hive cells; wherein a higher amount of the set of hive cells that are emptied is associated with a greater degree of hygienic behavior. In further embodiments, the set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the emptied hive cells are capped hive cells or uncapped hive cells.

In other embodiments, the presently disclosed subject matter is directed to a method for assessing the degree of hygienic behavior within a honey bee colony comprising a) applying a tritriacontene composition to a set of capped hive cells; b) exposing the set of capped hive cells to a honey bee colony; and c) determining the amount of uncapped and/or recapped hive cells in the set of capped hive cells; wherein a higher amount of the set of hive cells that are uncapped and/or recapped is associated with a greater degree of hygienic behavior. In further embodiments, the set of capped hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the set of capped hive cells is empty.

In other embodiments, the presently disclosed subject matter is directed to a method for assessing the degree of hygienic behavior within a honey bee colony comprising a) applying a mite-infested brood extract composition to a set of capped hive cells; b) exposing the set of capped hive cells to a honey bee colony; and c) determining the amount of uncapped and/or recapped hive cells in the set of capped hive cells; wherein a higher amount of the set of hive cells that are uncapped and/or recapped is associated with a greater degree of hygienic behavior. In further embodiments, the set of capped hive cells contains a diseased brood, diseased honey bees, or pests or parasites. In another embodiment, the set of capped hive cells is empty.

In further embodiments, the tritriacontene composition, the mite-infested brood extract composition, the diseased brood or diseased honey bees, pests or parasites, hive cells, emptied hive cells, hygienic behavior, and other terms may be as described herein.

V. EXAMPLES 1. Behavioral Study

Methods

Overview

Over two consecutive summers, sections of honey bee frames containing eggs from queens of various breeds, representing various levels of hygienic behavior, were grafted together. Grafted frames were placed into novel hives for rearing (such that no egg went back into its hive of origin). Varroa mite, wound and control treatments were applied. Removal status of each brood cell over a one week period was recorded. This experimental setup allowed comparison of removal 1) between brood types within each hive type, and 2) for each brood type across hive types.

Materials

Wooden frames, wax foundation, and unselected control (CON) queens were purchased from Triad Bee Supply in Trinity, N.C. Minnesota Hygienic (HYG) queens were donated by Jeff Hull and Amy Weeks in West Monroe, La. Varroa Sensitive Hygienic (VSH) queens were donated by the United States Department of Agriculture's Agricultural Research Center (USDA-ARC) in Baton Rouge, La. All queens were open mated and studied for one bee season only. Sample sizes for hives of CON, HYG and VSH origin were 2, 4 and 3, respectively for 2013, and 2, 2 and 2, respectively for 2014. Directly following the behavioral experiments, freeze-killed brood (FKB) assays were performed to determine the level of hygienic behavior exhibited by each hive. [100] However, due to missing queens and/or insufficient brood frames, successful results were only obtained for 11 of the 15 hives tested.

Methods

All behavioral assays were conducted at the University of North Carolina at Greensboro bee-yard during the summers of 2013 and 2014. Each year, medium wooden frames were sawed vertically into equal thirds and reassembled using metal brackets or staples. Reassembled frames were fitted with new wax foundation containing steel vertical wire stabilizers. Frames were placed into the top box of unselected CON hives, above queen excluders, so that the comb could be drawn out but the queen could not lay eggs in the cells.

Once combs were drawn out, frames were removed from unselected hives and fitted with wire cages constructed using mesh size 0.5 cm×0.5 cm, such that workers could move freely on and off both sides of each frame but queens, once introduced, could not leave. Single queens of unselected control (CON), Minnesota Hygienic (HYG), and Varroa-Sensitive Hygienic (VSH) breeds were placed on each frame. Caged experimental frames were returned to each queen's respective hive. Once eggs were present on >75% of both sides of a frame, frames were removed from their hives. A razor blade was used to cut combs into thirds (corresponding to the previous frame cuts) and metal brackets and staples were removed. Sections from different frames were then grafted together using metal brackets and staples such that each new frame contained eggs from different queens of VSH, HYG or CON origin. Frames were then redistributed into new hives, such that no brood was ever placed back into its hive of origin.

After allowing 5-7 days for development, the location of uncapped cells containing 5^(th) larval instars were marked using a permanent marker and transparent plastic sheet secured over each frame with thumb tacks. Cells along wires were avoided since wires are associated with increased brood removal rates. Frames were returned to their hives for 12 to 16 hours and then checked to ensure that they had been sealed with a wax cap for the initiation of pupation. This procedure ensured that treatments were administered to capped cells within 18 hours of capping, as is necessary to ensure initiation of mite oogenesis. [165] Mite, wound and control treatments were administered to recently capped cells in each section of frame corresponding to a different breed. To open the caps of experimental cells, one side of the cap was cut with the edge of a razor blade. The cell cap could then be lifted up, and resealed after treatment administration by pressing the cell cap against the cell wall with the edge of the razor. The treatments assigned to each cell were selected at random, and each cell received either one mite, one wound, or the control treatment. Mites were collected from non-experimental colonies at the UNCG bee-yard using the sugar shake method. [105, 187] Mites were shaken on to a damp paper towel, and were gently rinsed with a drop of clean water before being introduced to cells using a fine-tipped paintbrush within approximately one hour of collection. Mites that could not clutch the paintbrush bristles were considered to be unhealthy, and were not used. Wounds were inflicted using 50 m diameter capillary needles on the dorsal side of the brood between the first abdominal segment and the second thoracic segment according to existing protocols that mimic Varroa mite feeding [186, 35] Control cells were opened and resealed just as mite and wound cells, but received neither mite nor wound treatment. In 2013, a total of 1,063 cells were included in the study (349 VSH, 462 HYG, and 252 CON), and in 2014, an additional 1,025 cells were included (320 VSH, 354 HYG, 351 CON).

Each day for one week following treatment administration, experimental frames were removed from their hives for no more than 30 minutes per day to allow monitoring of each experimental cell for uncapping and removal. Brood removed on the first day following treatment introductions was excluded to avoid experimental artifacts, such as removal triggered by poorly resealed cells.

Statistical Analysis

A full factorial logistic regression model was used to determine the effects of brood type, hive type, treatment and year on brood removal. This analysis revealed higher order interactions (see results) that made the full data set difficult to interpret. Thus, the data set was split and Chi-square tests were used to determine the effect of brood type on removal (the focus of our hypothesis) for each treatment by hive type by year combination. Bonferroni correction was used for pairwise comparisons to control the family-wise error rate within each treatment by hive type by year combination (using a corrected significance threshold of p≤0.0167). Overall effects of brood type, hive type and treatment were assessed independently of each other using separate Chi-square analyses for each year. For the 11 hives with FKB assay data, Pearson product-moment correlation coefficients were computed to assess the relationships between 1) average percentage removal of brood in relation to the level of hygiene of the brood's hive of origin (overall and for each treatment type: mite, wound, or control), and 2) average percentage removal of brood in relation to the level of hygiene of its host hive (overall and for each treatment type: mite, wound, or control). All statistical analyses were performed using IBM SPSS Statistics, Version 22.

Results

Effects of Brood Type, Hive Type and Treatment on Removal Rates

The full logistic regression model, including the effects of brood type, hive type, treatment, year and all interactions on brood removal, was statistically significant (χ²=328.1, df=53, p<0.001)). The model achieved a correct classification of brood removal in 83.3% of the cases with an associated Nagelkerke's R² of 0.24. Multiple factors and interactions were statistically significant. (Table 1).

TABLE 1 Statistically factors and interactions from a full factorial logistic regression model on hive type, brood type, treatment and year. 95% C.I. for EXP(B) Factor Wald d.f. Sig. EXP(B) Lower Upper VSH Hive 5.436 1 0.020 0.072 0.008 0.658 HYG Brood 6.699 1 0.010 0.162 0.041 0.643 VSH Brood 7.165 1 0.007 0.098 0.018 0.537 Year 10.914  1 0.001 0.025 0.003 0.225 HYG Brood × 8.739 1 0.003 44.045  3.581 541.087  VSH Hive VSH Brood × 4.365 1 0.037 20.364  1.205 344.068  VSH Hive HYG Brood × 4.996 1 0.025 7.771 1.287 46.922  Mite Treatment VSH Brood × 8.482 1 0.004 64.684  3.910 1070.046   Year VSH Brood × 4.211 1 0.040 0.008 <0.001  0.807 VSH Hive × Year

In order to understand our results in detail, the effects of brood type on removal were evaluated for each treatment by hive type by year combination. These analyses revealed the importance of brood type on removal in most contexts when the brood was injured or mite parasitized, although effects varied between years (FIG. 1). To evaluate the overall effect of each individual factor (treatment, hive type, and brood type) on brood removal regardless of the other factors, data were pooled across factors to study average effects of one factor at a time. Chi-square analyses were used to determine the individual effects of treatment, hive type and brood type on removal for each year.

Treatment had a significant effect on brood removal rate in 2013 and 2014 (2013: χ²=31.3, d.f.=2, p<0.001; 2014: χ²=73.4, d.f.=2, p<0.001) (FIG. 2). In 2013, removal of mite-infested brood was not significantly higher than removal of wounded brood (χ²=2.4, d.f.=1, p=0.125) but was significantly higher than removal of control brood (χ²=31.2, d.f.=1, p<0.001). Removal of wounded brood was significantly higher than removal of control brood in 2013 (χ²=16.6, d.f.=1, p<0.001). In 2014 removal of mite-infested brood was significantly higher than removal of wounded brood (χ²=30.3, d.f.=1, p<0.001) and control brood (χ²-61.9, d.f.=1, p<0.001). Removal of wounded brood was significantly higher than removal of control brood in 2014 (χ²=7.4, d.f.=1, p=0.006).

Hive type had a significant effect on brood removal rate in 2013 but not in 2014 (2013: χ²=61.1, d.f.=2, p<0.001; 2014: χ²=2.7, d.f=2, p=0.263) (FIG. 3). In 2013, removal of brood in VSH hives was significantly higher than removal of brood in HYG hives (χ²=57.5, d.f.=1, p<0.001), but not significantly different than removal of brood in CON hives (χ²=0.8, d.f.=1, p=0.359). Removal of brood in HYG hives was significantly lower than removal of brood in CON hives in 2013 (χ²=37.8, d.f.=1, p<0.001). In 2014, there was no significant difference between removal in CON and HYG hives (χ²=2.6, d.f.=1, p=0.104), CON and VSH hives (χ²=0.5, d.f.=1, p=0.480), or HYG and VSH hives (χ²=0.8, d.f.=1, p=0.378).

In the subset of data for which hive type was the same as brood type, hive type had a significant effect on brood removal rate in 2013 and 2014 (2013: χ²=28.3, d.f.=2, p<0.001; 2014: χ²=10.5, d.f.=2, p=0.005) (FIG. 4). In 2013, removal of brood in VSH hives was significantly higher than removal of brood in HYG hives (χ²=5.26, d.f.=1, p=0.022), and significantly lower than removal of brood in CON hives (χ²=11.269, d.f.=1, p=0.001). Removal of brood in HYG hives was significantly lower than removal of brood in CON hives in 2013 (χ²=28.602, d.f.=1, p<0.001). In 2014, removal of brood in VSH hives was significantly higher than removal of brood in CON hives (χ²=10.548, d.f.=1, p=0.001), but was not significantly different from removal of brood in HYG hives (χ²=1.973, d.f.=1, p=0.160). Removal of brood in HYG hives was not significantly different than removal of brood in CON hives in 2014 (χ²=2.594, d.f.=1, p=0.107).

Brood type had a significant effect on brood removal rate in 2013 and 2014 (2013: χ²=31.4, d.f.=2, p<0.001; 2014: χ²=⁴2.1, d.f.=2, p<0.001) (FIG. 5). In 2013, removal of VSH brood was significantly lower than removal of HYG brood (χ²=27.6, d.f.=1, p<0.001) but not significantly different than removal of CON brood (χ²=2.0, d.f.=1, p=0.155). Removal of HYG brood was significantly higher than removal of CON brood in 2013 (χ²=12.9, d.f.=1, p<0.001). In 2014, removal of VSH brood was significantly higher than removal of HYG brood (χ²=15.2, d.f.=1, p<0.001) and CON brood (χ²=38.3, d.f.=1, p<0.001). Removal of HYG brood was not significantly different than removal of CON brood in 2014 (χ²=2.3, d.f.=1, p=0.125).

Effects of Hygiene Level on Removal Rate

A significant, positive correlation was identified between removal of brood (regardless of hive breed and treatment) and the level of hygiene of the brood's hive of origin (r=0.680, n=1, p=0.021). No comparable correlation was identified between removal of brood and the level of hygiene of the brood's host hive across treatments and brood breeds (r=0.414, n=1, p=0.205). There was a significant positive correlation between removal of brood and the level of hygiene of the brood's hive of origin for the mite treatment (r=0.658, n=1, p=0.028; FIG. 6A, Line A). No significant correlation was found for wound (r=0.563, n=11, p=0.072) or control (r=0.455, n=11, p=0.160) treatments (FIG. 6A, Lines B and C, respectively). There was a significant negative correlation between removal of brood and the level of hygiene of the host hive for the wound treatment (r=−0.766, n=11, p=0.006; FIG. 6B, Line B). No significant correlation was found for mite (r=−0.134, n=11, p=0.694) or control (r=−0.389, n=11, p=0.237) treatments (FIG. 6B, Lines A and C, respectively). FIGS. 6A and 6B do not include hives without % hygienic data (n=4).

2. Chemical Study Methods a. Experiment 2-1

Sample Collection

This chemical study analyzed how cuticular profiles of Varroa-Sensitive Hygienic (VSH), Minnesota Hygienic (HYG) and unselected control (CON) honey bee brood of varying Varroa mite resistance are influenced by Varroa mite, wound and control treatments. All sample collection and analysis was conducted at the University of North Carolina at Greensboro during the summers of 2012, 2013 and 2014. Queens of VSH (n=6, 2 each in 2012, 2013 and 2014), HYG (n=8, 4 in 2012, and 2 each in 2013 and 2014), and control (CON) (n=6, 4 in 2012 and 2 in 2014) origin were caged on wax foundation frames (1 queen per frame). All frames had been drawn out in control (CON) hives immediately before the experiment and were inserted into to each queen's respective hive. In 2013, control (CON) queens did not perform as expected and therefore no data are available from that year for this group. Queen cages were removed once eggs were laid in greater than 75% of cells. After allowing 5 to 6 days for larval development, the locations of uncapped cells containing 5^(th) instar larvae were marked using transparent plastic sheets held in place above the experimental cells with thumb tacks. Frames were placed back in the hives for no more than 16 hours to be capped. These recently capped cells were used to ensure that the experimental treatments were all applied to larvae at the same age, within 16 hours of capping. This time window is critical for successful initiation of mite oogenesis. [165]

Within 16 hours of cell capping, mite, wound and control treatments were introduced into marked, capped cells in each frame, and the location of cells containing each treatment was marked on the transparent plastic sheets. Mites used were in the phoretic stage, collected from adult worker bees from control (CON) hives by sugar shake. [105, 187] The collected mites, were introduced to cells using a fine-tipped paintbrush within approximately one hour of collection. Wounds were inflicted using 50 μm diameter capillary needles that mimic mite-inflicted feeding sites [35] and were administered on the dorsal side of the brood between the first abdominal segment and the second thoracic segment according to existing protocols. [186] Control cells were opened and resealed just as mite and wound cells, but received neither mite nor wound treatment. Frames were returned to their hive of origin for 24 hours to allow bees to reseal the cell caps and were then transferred to an incubator maintained at 34° C. with 50% relative humidity (RH). One to four brood from each breed-by-treatment group were collected each day on days 4, 5, and 6 post-capping. These days were chosen because brood removal rates were highest on these three days in a preliminary behavioral study (data not shown). Each individual brood was placed inside a 2 mL screw top glass vial with silicone septa (Agilent, product number 5190-2278). Extraction of brood cuticular chemicals was performed within one hour of sample collection, and no brood that appeared to be damaged from the collection was used.

Cuticular Chemistry

Individual brood were submerged and soaked in hexane for 9 minutes in order to collect non-polar cuticular compounds. The volume of hexane used was varied between approximately 0.5 mL and 1.5 mL, depending on the volume needed to completely submerge each sample. After 9 minutes, the hexane extract was removed from the brood and stored in a separate 2 mL glass vial. Brood and hexane samples were stored at −80° C. until analysis. For extract analysis, hexane was evaporated overnight under a Fisher Hamilton SAFEAIRE® hood. Samples were reconstituted with 50 μL of heptane the following day, after complete evaporation. Heptane used for reconstitution was spiked with butyl butyrate (1 μL butyl butyrate per 10 mL heptane) as an internal standard. For reconstitution, heptane was added to each sample vial for 3 minutes, and the resulting sample transferred into a 400 μL glass flat bottom insert (Agilent product number 5181-3377) using a gas tight 100 μL syringe (Hamilton product number 81062). Glass inserts were used to facilitate operation of the Gas Chromotography Mass Spectrometry (GCMS) autosampler. Use of heptane rather than hexane prevented evaporation of the sample during operation of the autosampler. Samples were analyzed by GCMS to characterize qualitative and quantitative features of the chemical brood extracts. Samples were analyzed at the University of North Carolina at Greensboro on a Shimadzu GCMS-QP2010S (operating at 0.97 kV and acquiring m/z values from 40 to 650). Source and interface temperatures were 230° C. and 250° C. respectively. A 30 m ZebronZB-5MS column with 0.25-mm diameter, 0.5-μm stationary phase thickness was used with helium as the carrier gas (column head pressure 70.2 kPa, total flow rate of 18.1 ml/min, column flow rate of 1.05 ml/min, linear velocity 37.8 cm/sec, purge flow 0.5 mL/min, split ratio −1.0). Column oven temperature was 80° C., injection temperature was 280° C. and injection mode was splitless. After a 1 minute hold, the oven temperature rose from 80 to 165° C. at 15° C./min, and then from 165 to 320° C. at 10° C./min, with a final hold at 320° C. for 10 minutes.

Qualitative and quantitative data were collected for individual honey bee brood samples using GCMS. Since novel peaks were not expected, only the internal standard and cuticular chemicals (n=33) that were reproducibly quantifiable between the majority of samples were used for analysis. For qualitative analysis (peak identification) we used the mass spectral libraries NIST 2005 and WILEY 2007, including supplementary editions. GC-MS post-run analysis software calculated match percentage using an algorithm that compared spectra of the compounds of interest with ions from known library spectra. The length of saturated hydrocarbons was confirmed based on comparison with an external standard composed of Supelco n-hydrocarbon mix (even-numbered alkanes from C8 to C40, diluted 1000:1 with heptane) and spiked with pentadecane (C15). For quantitative analysis, we calculated the proportion of each chemical relative to the total chemicals measured. For each brood sample, we divided the area under each peak (individual peak area) by the sum of the area under all 34 peaks of interest (total peak area), including the 33 cuticular chemicals of interest and the internal standard butyl butyrate. Using the equation below, arcsin transformation was performed for normalization of the proportion data and stabilization of variance. [188]

${RQ} = {{\sin^{- 1}\left( \sqrt{\frac{{individual}\mspace{14mu} {peak}\mspace{14mu} {area}}{\sum{{total}\mspace{14mu} {peak}\mspace{14mu} {areas}}}} \right)} \times (100)}$

Triplicate runs for n=2 samples indicated that error due to auto-sampler injection was minimal (average standard error for peak area between triplicate samples was ±0.002). Therefore repeated injections were deemed unnecessary, and each sample for the main analysis was run only one time.

Virus Quantification

After hexane extraction, the quantity of Deformed Wing Virus (DWV) for individual honey bee brood was analyzed. For each sample, RNA was extracted, cDNA was synthesized, and quantitative PCR (qPCR) was performed. For RNA extraction, brood were transferred from glass vials to 2 mL Eppendorf® tubes and homogenized with 0.5 mL TRIzol (ambion by Life Technologies) using a plastic pestle. Samples were incubated at room temperature for 10 minutes. After incubation, another 0.5 mL TRIzol was added to each sample, and then samples were vortexed for twenty seconds on the highest speed setting (#10) of a Fisher Scientific Mini Vortexer. Next, 0.2 mL chloroform was added to each sample, and samples were vortexed again. Samples were incubated at room temperature for 3 minutes, and then centrifuged at 12,000 RCF for 15 minutes. The top layer of each sample was then pipetted into a 1.5 mL tube containing 0.5 mL of isopropanol. Samples were mixed and placed on ice for 15 minutes, and then centrifuged again at 12,000 RCF for 10 minutes. The supernatant was discarded, and 1 mL of 75% ethanol was added to each pellet, before centrifuging again at 7,500 RCF for 5 minutes. The superantant was discarded again, and samples were allowed to air dry for 15 minutes. Next, 0.1 mL of molecular grade water (G Biosciences cat#786-72C) was added to each sample to resuspend the pellets. Samples were stored at −80° C. until used for cDNA synthesis.

To determine sample concentration and purity of the RNA extract for cDNA synthesis, a 1 μL RNA aliquot from each extract was analyzed using a Nanodrop ND-1000 Spectrophotometer. The amount of sample needed for 2,000 ng of RNA was then calculated and pipetted into 1.5 mL Eppendorf® tubes. Water was added such that each sample reached a total volume of 8 μL, and then 2.2 μL of DNAse solution containing 1 μL of DNAse 1 μL of DNAse buffer and 0.2 μL of RNAse Out was added to each sample. Samples were then heated to 37° C. for one hour, and then to 75° C. for 10 minutes. Next, for 2013 samples only, 1 μL of a solution containing 0.02 μL dT, 0.5 μL random hexamer, 0.2 μL dNTP, and 0.298 μL H2O was added to each sample. Samples were incubated at 65° C. for 5 minutes and then chilled on ice for 10 minutes. Next, for 2013 samples, 10 μL of a Master Mix containing 4 μL of First Strand buffer, 2 μL of DTT, 0.5 μL of Super Scriptase II, and 3.5 μL of molecular grade water was added to each sample. For 2014 samples, 9.8 μL of Master Mix containing 4 μL 5× TransAmp Buffer, 1 μL Reverse Transcriptase and 4.8 μL of molecular grade water was added to each sample. Samples were incubated at 42° C. for 50 minutes, and then 70° C. for 15 minutes. Samples were stored at −20° C. until used for RT-qPCR analysis.

RT-qPCR was performed to determine the quantity of DWV in each sample. For each 2013 sample, 1 μL of cDNA, 10 μL Power SYBR Green Mix, 8 μL of water, 0.5 μL of forward primer (sequence: 5′-GAGAUTGAAGCGCATGAACA-3′)(SEQ ID NO. 1), and 0.5 μL of reverse primer (sequence: 5′-TGAATTCAGTGTCGCCCATA-3′)(SEQ ID NO. 2) were added to 0.1 ml MicroAmp Fast Optical 96-Well Reaction Plate tubes. For each 2014 sample, 2 μL of cDNA, 10 μL of 2× Sensifast SYBR Hi-ROX Mix, 7.2 μL of water, 0.4 μL of forward primer, and 0.4 μL of reverse primer were added to 0.1 ml MicroAmp Fast Optical 96-Well Reaction Plate tubes. Liquid was centrifuged to the bottom and samples were run through 45 cycles on an Applied Biosystems StepOne Plus qPCR machine set to SYBR as the passive agent. Samples were analyzed for DWV as well as for the two reference genes. Actin (forward primer sequence: 5′-TTGTATGCCAACACTGTCCTTT-3′ (SEQ ID NO. 3); reverse primer sequence: TGGCGCGATGATCTTAATTT-3′ (SEQ ID NO. 4)) and RPS5 (forward primer sequence: 5′-AATTATTTGGTCGCTGGAATTG-3′ (SEQ ID NO. 5); reverse primer sequence: 5′-TAACGTCCAGCAGAATGTGGTA-3′ (SEQ ID NO. 6)). Each transcript for each sample was run in triplicate.

Based on RT-qPCR results, DWV was classified as either “low” or “high” for each sample. Samples were categorized as “low” for DWV if the cycle threshold (C_(T)) was undetermined in all three replicates, or if only one replicate contained a determined C_(T) with a secondary peak at the correct melting temperature (Tm). Samples were categorized as “high” for DWV if at least one replicate contained a determined C_(T) with the primary peak at the correct Tm, or if two or more replicates contained determined C_(T) values with a secondary peaks at the correct Tm. DWV was also placed on a continuous scale by calculating an average Delta C_(T) for each sample. Delta C_(T) was calculated for each sample by taking the average of the Delta C_(T) across all three replicates. When no C_(T) value was determined, a C_(T) of 45 (the number of cycles used for RT-qPCR) was used. Delta C_(T) was calculated using the following equation, such that the higher the Delta C_(T) value, the greater the amount of DWV in the sample:

Delta CT=CT(Actin)−CT(DWV)

Statistical Analysis

A full factorial MANOVA was used to determine whether treatment (mite, wound and control) affected chemical signatures of brood across brood type (control (CON), HYG and VSH) and age (days 4, 5, and 6 post-capping) age. The Wilks' Lambda statistic is reported. As a follow-up to MANOVA, ANOVAs were used to evaluate how certain chemicals of interest differed by treatment for each brood type. ANOVA was also used to determine the effects of DWV load on mean P32 quantity of brood. A significance level of 0.05 was used for all statistical tests. All statistical analyses were performed using IBM SPSS Statistics, Version 22.

b. Experiment 2-2

Sample Collection

A combined behavioral-chemical study analyzed how cuticular profiles of uncapped, mite infested honey bee brood differed from those of control brood that remained capped. As described above, mite and control treatments were introduced to recently capped cells in single frames from each of two VSH hives. The cells containing introduced mites were monitored periodically for uncapping from day 3 to day 6 post-capping. Each time one mite-infested brood was found to be uncapped and apparently unharmed (ie: removal by nurse bees had not yet begun), the brood was collected. Each time an uncapped, mite-infested brood was collected, two control brood were also collected at the same time—one from a capped, mite-infested cell, and one from a capped control cell (FIG. 9A). In total, six uncapped, mite-infested cells were collected, three from each hive. In the first hive, the 3 uncapped brood and their 6 controls were collected on days 3, 3, and 6 post-capping. In the second hive, the 3 uncapped brood and their six controls were collected on days 5, 6 and 6 post-capping. Each individual brood was placed inside a 2 mL screw top glass vial. Extraction of brood cuticular chemicals was performed within one hour of sample collection as described above, ensuring by visual inspection that no brood appeared to be damaged from the collection.

Cuticular Chemistry

As described above for Experiment 2-1, individual brood were submerged and soaked in hexane for 9 minutes in order to collect non-polar cuticular compounds. All methodology for chemical extraction and analysis in Experiment 2-2 was the same as that listed above for Experiment 2-1. However for each sample in Experiment 2-2, only the relative quantity of the six peaks that were found to differ significantly with treatment in Experiment 2-1 (P32, P28, P25, P18, P15, and P14) were evaluated.

FIG. 7 shows the GCMS output from a single, mite-infested honey bee, illustrating the location of the P32 peak (top) and the mass-to-charge ratio for P32 (bottom).

Virus Quantification

As described above for Experiment 2-1, individual brood were analyzed for DWV content. All methodology for RNA extraction, cDNA synthesis, RT-qPCR, and RT-qPCR analysis in Experiment 2-2 was the same as that listed for 2014 in Experiment 2-1.

Statistical Analysis

Paired-sample t-tests were used to determine whether the mean quantity of each of the six peaks identified to be significantly affected by treatment in Experiment 2-1 differed for the following paired samples: uncapped, mite-infested honey bee brood vs. capped mite-infested brood; uncapped, mite-infested honey bee brood vs. capped mite-free controls; and capped mite-infested brood vs. capped mite-free controls. Paired-sample t-tests were chosen because uncapped samples were collected on different times and days. Because honey bee brood chemical are age dependent, samples could only be compared with those collected at the same time point. Since direction of the differences could be predicted before the tests based on results from Experiment 2-1, a one-tailed statistical test was used. A significance level of 0.05 was used for all statistical tests.

Results a. Experiment 2-1

A full factorial MANOVA was run to understand the effects of treatment, brood type, age, and their interactions on the quantities of the 33 honey bee cuticular chemicals. Significant effects on brood cuticular profiles were identified for treatment (F=1.61, d.f.=66,708, p=0.002), brood type (F=5.42, d.f.=66.708, p<0.001), and age (F=26.52, d.f.=66,708, p<0.001). A treatment-by-age interaction (F=1.27, d.f.=132, 1411, p=0.026), and a brood type-by-age interaction (F=1.59, d.f.=132, 1411, p<0.001) were also found. No significant effects were identified for the treatment-by-brood type interaction (F=0.74, d.f.=132, 1411, p=0.987) or the treatment-by-brood type-by-age interaction (F=0.53, d.f.=264,2775, p=0.877).

Two-way ANOVAs indicated that of the 33 chemicals analyzed, 6 were significant for treatment effects, 18 were significant for brood type effects, 29 were significant for age effects, 1 was significant for treatment-by-age interaction effects, and 12 were significant for brood type-by-age interaction effects. Of the 6 chemicals significantly affected by treatment, the mean quantity of peak number 32 (P32) was found to be higher in mite-infested brood than in controls while the mean quantity of peaks numbered 28, 25, 18, 15, and 14 (P28, P25, P18, P15 and P14, respectively) were found to be lower in mite-infested brood than in controls (data not shown). Since the primary interest of this study was to explore the effects of interactions between treatment and brood type on cuticular chemicals, the 6 peaks significantly affected by treatment were explored in more depth using separate ANOVAs for each brood type. While the mean quantity of P32 was higher in mite-infested brood than control brood for all three brood breeds, the ANOVAs indicated that this difference was only significant for VSH brood (F=42.67, d.f.=1, p=0.031), and was not significant for HYG (F=1.06, d.f.=1, p=0.307) or control (CON) (F=0.10, d.f.=1, p=0.760) brood (FIG. 8, top row; sample size: control (CON) brood n=18 for both treatments; HYG brood n=41 for control and n=42 for mite-treated; VSH brood n=44 for control and n=39 for mite-treated). Since the effects of brood breed and virus levels on cuticular chemicals were also of interest, the effect of DWV on P32 was also explored using separate ANOVAs for each brood type. The virus data from 2012 brood was incomplete and much higher than that of the following years. We deemed those 2012 values unreliable and did not include them in the analysis. Thus, all virus data refers to brood from 2013 and 2014. The mean quantity of P32 was significantly higher in brood with high DWV levels than in those with low DWV levels for both control (CON) (F=6.52, d.f.=1, p=0.014) and HYG (F=12.45, d.f.=1, p=0.001) brood (FIG. 8, bottom row; sample size: control (CON) brood n=32 for low DWV levels and n=22 for high DWV levels; HYG brood n=96 for low DWV levels and n=30 for high DWV levels; VSH brood n=62 for low DWV levels and n=62 for high DWV levels). There was no significant effect of DWV level on P32 for VSH brood (F=0.17, d.f.=1, p=0.679). For the sake of comparison between the effects of treatment and virus levels on P32, results for the effect of treatment on the mean quantity of P32 for each brood type were also calculated using only the data from 2013 and 2014. As with the full data set, the mean quantity of P32 was higher in mite-infested brood than control brood for all three brood breeds, but the ANOVAs indicated that this difference was only significant for VSH brood (F=4.26, d.f.=2, p=0.016), and not for HYG (F=0.863, d.f.=2, p=0.424) or control (CON) (F=0.069, d.f.=2, p=0.933) brood.

Individual ANOVAs suggested that the mean quantities of P28, P25, P18, P15 and P14 were not significantly different between treatment groups in any of the three brood types (data not shown). The mean quantity of P28 was significantly lower in brood with high DWV levels than in those with low DWV levels for both control (CON) (F=13.31, d.f.=1, p=0.001) and HYG (F=14.90, d.f=1, p<0.001) brood. There was no significant effect of DWV level on P28 for VSH brood (F=1.93, d.f.=1, p=0.167). The mean quantity of P25 was significantly lower in brood with high DWV levels than in those with low DWV levels for both control (CON) (F=11.32, d.f.=1, p=0.001) and HYG (F=8.45, d.f.=1, p=0.004) brood. There was a suggestive effect of DWV level on P25 for VSH brood (F=3.00, d.f.=1, p=0.086). The mean quantities of P18, P15 and P14 were not significantly different for DWV levels in any of the three brood types (data not shown).

b. Experiment 2-2

For each of the six peaks identified (P32, P28, P25, P18, P15, and P14) to be significantly affected by treatment in Experiment 2-1, paired-sample t-tests were used to compare the mean peak quantity of uncapped, mite-infested brood to either capped, mite-infested brood or capped mite-free controls. Paired-sample t-tests were also used to compare mean peak quantity of capped, mite-infested brood and capped, mite-free controls. Mean P32 quantity was significantly higher for uncapped, mite-infested brood than for either capped mite-infested (t=2.85, df=5, p=0.018) or capped mite-free (t=3.71, df=5, p=0.007) controls (FIG. 9B). Each relative quantity of P32 is based on an average of six samples. Each time collection from a mite-infested cell that had been uncapped was made (n=6), collection from two controls were also made, one with a mite (n=6) and one without (n=6).) As shown in FIG. 9B, there was no significant different in mean P32 quantity between capped mite-infested and capped mite-free controls (t=1.24, df=5, p=0.136).

Mean P28 quantity was significantly lower for uncapped, mite-infested brood than for the capped mite-free (t=2.17, df=5, p=0.041) control. There was no significant difference in mean P28 quantity between capped mite-infested and either the uncapped, mite-infested brood (t=1.16, df=5, p=0.150) or the capped mite-free control (t=0.99, df=5, p=0.184). Mean P25 quantity was not significantly different for uncapped, mite-infested honey bee brood and capped mite-infested brood (t=−0.63, df=5, p=0.279), uncapped, mite-infested honey bee brood and capped mite-free controls (t=−0.53, df=5, p=0.310), or capped mite-infested brood and capped mite-free controls (t=−0.10, df=5, p=0.464). Mean P18 quantity was not significantly different for uncapped, mite-infested honey bee brood and capped mite-infested brood (t=−1.42, df=5, p=0.108), uncapped, mite-infested honey bee brood and capped mite-free controls (t=−1.40, df=5, p=0.111), or capped mite-infested brood and capped mite-free controls (t=−0.84, df=5, p=0.220). Mean P15 quantity was not significantly different for uncapped, mite-infested honey bee brood and capped mite-infested brood (t=−0.14, df=5, p=0.449), uncapped, mite-infested honey bee brood and capped mite-free controls (t=1.00, df=5, p=0.182), or capped mite-infested brood and capped mite-free controls (t=1.00, df=5, p=0.182). Mean P14 quantity was not significantly different for uncapped, mite-infested honey bee brood and capped mite-infested brood (t=−1.64, df=5, p=0.082), uncapped, mite-infested honey bee brood and capped mite-free controls (t=−0.84, df=5, p=0.221), or capped mite-infested brood and capped mite-free controls (t=0.61, df=5, p=0.284). DWV was only detected in one sample: a capped, mite-infested control, so DWV is not thought to play a role in differences measured.

Consistent results were obtained with an increased sample size, wherein each relative quantity of P32 is based on an average of 24 samples (the previously analyzed 6 samples from 2014 and 18 samples from 2015). Each time collection from a mite-infested cell that had been uncapped was made (n=24), collection from two controls were also made, one with a mite (n=24) and one without (n=24). ANOVA was used to compare the mean peak quantity of uncapped, mite-infested brood to either capped, mite-infested brood or capped, mite-free controls. ANOVA was also used to compare mean peak quantity of capped, mite-infested brood and capped, mite-free controls. As shown in FIG. 9C, uncapped mite-infested brood had a significantly higher mean relative percent P32 than did capped mite-infested (F_((1,46,))=16.956, p<0.001) or capped control (F_((1,46))=21.429, p<0.001) brood. There was no significant difference between the mean relative percent P32 for capped mite-infested and capped control brood (F_((1,46))=0.654, p=0.423).

Mean P28 quantity was not significantly different for uncapped, mite-infested honey bee brood and capped mite-infested brood (F_((1,46,))=0.508, p=0.479), uncapped, mite-infested honey bee brood and capped mite-free controls (F_((1,46,))=0.310, p=0.580), or capped mite-infested brood and capped mite-free controls (F_((1,46,))=0.017, p=0.898). Mean P25 quantity was not significantly different for uncapped, mite-infested honey bee brood and capped mite-infested brood (F_((1,46,))=0.073, p=0.788), uncapped, mite-infested honey bee brood and capped mite-free controls (F_((1,46,))=0.784, p=0.380), or capped mite-infested brood and capped mite-free controls (F_((1,46,))=0.304, p=0.584). Mean P18 quantity was not significantly different for uncapped, mite-infested honey bee brood and capped mite-infested brood (F_((1,46,))=1.045, p=0.312), uncapped, mite-infested honey bee brood and capped mite-free controls (F_((1,46,))=0.296, p=0.589), or capped mite-infested brood and capped mite-free controls (F_((1,46,))=0.407, p=0.527). Mean P15 quantity was not significantly different for uncapped, mite-infested honey bee brood and capped mite-infested brood (F_((1,46,))=0.574, p=0.453), uncapped, mite-infested honey bee brood and capped mite-free controls (F_((1,46,))=0.127, p=0.723), or capped mite-infested brood and capped mite-free controls (F_((1,46,))=0.159, p=0.692). Mean P14 quantity was not significantly different for uncapped, mite-infested honey bee brood and capped mite-infested brood (F_((1,46,))=2.478, p=0.122), uncapped, mite-infested honey bee brood and capped mite-free controls (F_((1,46,))=0.939, p=0.338), or capped mite-infested brood and capped mite-free controls (F_((1,46,))=0.432, p=0.514). Again, DWV is not thought to play a role in differences in hygienic behavior measured. DWV titers were significantly higher in mite-infested cells than non-infested cells (F_((1,67))=3.005, p=0.044) but did not differ significantly between capped mite-infested cells and uncapped mite-infested cells (F_((1,46))=0.041, p=0.420).

3. Induced Hygienic Behavior Study with Mite-Infested Brood Extract

Methods

CHC extracts of the same volume but different concentrations representing 3 brood, 1 brood, and 0.3 brood (3, 1, 0.3 brood equivalents (BEqs) respectively) were transferred from mite-infested and control brood to the wax caps of healthy cells. The experiment could have been performed in any type of hive. Given the brood treatment effect described above, VSH hives were preferred and selected as a first test.

The bioassay was performed using experimental brood aged 4 days post-capping, as our previous findings suggest that this is the brood age at which hygienic behavior is most likely to occur (data not shown). Appropriately aged brood was identified 4 days before each bioassay via marking of recently capped cells, as described above.

To collect CHC extracts from brood, the location of uncapped cells containing 5^(th) instar larvae were marked using a permanent marker and transparent plastic sheet secured over each frame with thumb tacks. Frames were placed back into the hives. The following day, frames were recollected, and only brood that had been capped was marked for experimental use. Within 18 hours of capping, Varroa mites was introduced to approximately 50% of capped cells. Careful timing of Varroa introductions is necessary to ensure initiation of mite oogenesis. [165, 37] Mites used were collected by sugar shake [187, 105] from a donor colony and were introduced randomly to cells using a fine-tipped paintbrush. The remaining 50% of capped cells were control cells. Control cells were opened and resealed just as mite cells, but did not receive a mite.

Brood from 65 mite-infested cells and an equal number of non-infested cells were collected on the 4^(th) day post-capping. Pools of 5 brood were soaked for 9 minutes in approximately 1.5 ml hexane. The solvent containing brood cuticular extracts was then removed from the brood. The extracts collected from within each treatment group (mite-infested and control) were combined in their respective groups, evaporated overnight, and reconstituted using a total of 90 μl of hexane. After a 3 minute wait period the remaining 65 μl for each treatment group was aliquoted into vials labeled with three different brood-equivalent (BEq) concentrations: 3 BEq, 1 BEq, and 0.3 BEq. These vials received 45, 15 and 5 μl of the extract, respectively. Samples were evaporated overnight again, transported on ice to the bee station, and then reconstituted (one at a time) using 33 μl hexane. After a 3 minute wait period, the remaining 30 μl of extract was collected in an airtight syringe, and aliquoted onto 15 randomly selected wax caps of experimental cells (2 μl per cell). This was repeated for each extract concentration (3 BEq, 1 BEq and 0.3 BEq) for each treatment (mite and control). The brood frame was then placed back into the hive, and uncapping and removal were checked and recorded after 8, 24, and 48 hours.

A chi-square analysis was used to test for a difference in uncapping between treatment groups. The statistical test was performed using IBM SPSS Statistics, Version 22.

Results

Mite-infested brood extract at concentrations of 0.3 BEq and 1 BEq each induced more uncapping than control brood extract at concentrations of 0.3 BEq and 1 BEq, respectively; and more uncapping than hexane treatment (FIG. 10). Mite-infested brood extract at a concentration of 3 BEq did not demonstrate significant differences from control brood extract at a concentration of 3 BEq; however both demonstrate more uncapping compared to hexane treatment (FIG. 10). Control brood extract at a concentration of 3 BEq demonstrated uncapping effects, likely due to excessive concentrations of both hexane and control brood extract.

FIG. 11 compares control brood extract and mite-infested brood extract, using the same data represented in FIG. 10 and combining data sets from all three concentrations of 0.3 BEq, 1 BEq, and 3 BEq for each group. Mite-infested brood extract induced significantly more uncapping than extracts from control brood extract (FIG. 11) (χ²=3.64, d.f.=1, p=0.029).

While the majority of studies of hygienic behavior mechanisms have focused on sensitivity and modulation of adult olfaction [10, 31, 85, 166, 167, 175], recent evidence indicates the importance of brood signals in triggering of hygienic behavior. [102, 176, 179]

The chemical study above demonstrates significant P32 levels in i) Varroa mite-treated VSH brood (FIG. 8, top row), ii) both control and HYG brood with high levels of Deformed Wing Virus (FIG. 8, bottom row), and iii) uncapped hive cells with Varroa-infested brood (FIGS. 9B and 9C). The behavioral study above demonstrates a positive correlation between removal of brood and the level of hygiene of the brood's hive of origin.

4. Examples: Induced Hygienic Behavior Studies with a Tritriacontene

a. Synthesis of a Tritriacontene

Methods: Synthesis methods of a tritriacontene or agriculturally acceptable derivatives can be readily determined by those skilled in the art, including but not limited to the field of insect semiochemicals. Synthesis methods generally include, but are not limited to, alkylation of a long, straight-chained alkyne followed by reduction to form the double bond (see for example, use of two commercially available synthons [206]); a cis-selective Wittig reaction 12071; and an olefin metathesis reaction [205]. Also, certain tritriacontenes are commercially available. For example, (Z)-10-tritriacontene, which has the structure,

is commercially available (CAS Registry No. 99026-87-6). Products may be purified by gas chromatography and/or recrystallization. Identity and purity of the compound may be ascertained by GC-MS analysis. In addition to analyzing the synthesis product alone, it will be added to aliquots of hexane extracts of Varroa-mite infested VSH brood to quantify the increase of the P32 peak, relative to the other constituents of the extracted mixture.

Results:

(Z)-10-tritriacontene and (Z)-16-dotriacontene (Z10-C320), described below, were synthesized by collaborator Jocelyn Millar from the University of California Riverside using standard procedures. The identity of the synthesized (Z)-10-tritriacontene was verified using comparative GC-MS analysis of a honey bee brood cuticular extract with and without a spike of the synthesized compound. The peak from the synthesized (Z)-10-tritriacontene coincided perfectly with the P32 peak from the honey bee brood extract. Accordingly, “P32” and “(Z)-10-tritriacontene” and “Z10-C33” are used interchangeably herein.

b.i. Induced Hygienic Behavior Study with Synthesized P32

Methods:

The pure P32 compound will be aliquoted and diluted in hexane. 1 μl of different dilutions will be applied onto larvae and/or caps of individual brood cells in three experimental hives. The VSH, MH, and control genotype (together, hereinafter, referred to as “line”) will be tested for their response. Hives from each source will be set up in an experimental apiary and standardized in size, brood, and resources.

Brood frames containing uncapped 5^(th) larval instars will be collected from each hive, and the location of the uncapped cells will be marked using a plastic transparency [208]. Frames will be replaced into the hives, and cells that are capped within the next 16 hours will be recorded and used for the experiment. At day 4 post-capping, cells will be treated with hexane (a negative control) or one of the following dilutions of pure P32: 10⁻¹, 10⁻³, 10⁻⁵, 10⁻⁷, 10⁻⁹, 10⁻¹¹. Pin-killed brood will be used as a positive control. Twenty cells will receive each treatment in each colony for a total sample size of 1440 (20 cells×8 treatments×3 replicate hives×3 genotypes).

Treatments will be randomly distributed across experimental combs and the location of each treatment recorded on a clear transparent sheet put over the comb [208]. Cell uncapping behavior and removal of brood will be surveyed 1 hour, 4 hours, 8 hours, and 24 hours after treatment. Uncapping and removal rates at each time point will be compared among the different experimental groups in each colony. Chi-squared analysis will be used to test for differences in the frequency of hygienic behavior between treatment groups at each time point.

Results:

Synthesized P32 and controls were applied to cell caps by syringe. Results suggested significantly higher removal of P32-treated cells compared to hexane treated cells in both of the MH colonies tested. No VSH colonies were available for testing, and no significant difference was observed between removal of P32- and hexane-treated cells in control colonies. However, methodological issues justified the discarding of these results, and repetition of the experiment using improved methods. Specifically, chemical dilutions were not properly performed because the volume of the experimental chemical was not taken into account when making dilutions in the hexane solvent. Additionally, high variability between pin-killed treatments suggested that these were not reliable as positive controls. The following experiment serves as a replacement for the experiment described above. Improved methods in the replacement experiment below include corrected dilution procedures (solvent was added to the experimental chemical to a specific final volume), removal of the pin-killed positive control, and use of the alkene (Z)-16-dotriacontene (also called Z16-C32) as the negative control. Compared to the alkanes used previously, (Z)-16-dotriacontene more closely resembles P32, and thus serves as a more appropriate negative control. A new method for application of chemicals by airbrushing was also utilized to increase speed and quantity in chemical application (FIG. 12).

b.ii. Induced Hygienic Behavior Study with Synthesized P32 Applied to Cell Caps by Airbrushing

Methods:

The dose dependent removal effects of (Z)-10-tritriacontene and (Z)-16-dotriacontene applied to wax caps of honey bee brood cells were measured repeatedly in a single VSH colony. (Z)-10-tritriacontene, along with (Z)-16-dotriacontene and hexane controls were applied to capped honey bee brood cells using a Paasche H-100D Single Action Airbrush & Compressor system, operating at 25 PSI. To control the amount of brood cells exposed to each chemical treatment (and thus the dose per cell) 3″ diameter PVC pipe pieces were inserted into a brood frames containing capped pupae. Thus, for each treatment and replicate, 2 mL of solution was applied to a single 3″-diameter section of brood comb by airbrushing. Since there are roughly 200 cells per 3″ diameter circle, this is roughly 10 ul solution per cell. Concentrations of 1%, 0.3%, and 0.1% (Z)-10-tritriacontene and (Z)-16-dotriacontene were tested. Sample sizes for (Z)-10-tritriacontene were 532, 420, and 282 cells for concentrations of 1%, 0.3%, and 0.1%, respectively. Sample sizes for (Z)-16-dotriacontene were 289, 296, and 311 for concentrations of 1%, 0.3%, and 0.1%, respectively. Sample sizes for the hexane control and no treatment control were 425 and 154, respectively. The location of all assays on frames was randomized. The frequency of uncapping and removal was recorded at four hours and twenty-four hours post treatment. Chi-square analysis with Bonferroni correction was used to compare hygienic removal and uncapping between treatments.

Results:

Removal of pupae in brood cells treated with 1% and 0.3% (Z)-10-tritriacontene was significantly greater than that of pupae in all control brood cells, including cells treated with any concentration of (Z)-16-dotriacontene (FIG. 13). Both (Z)-10-tritriacontene and (Z)-16-dotriacontene appear to be removed in a dose-dependent manner, with the highest concentrations eliciting the greatest removal. These results indicate that the removal effect is specific to (Z)-10-tritriacontene, supporting our hypothesis that this (Z)-10-tritriacontene is a natural trigger of hygienic removal in honey bees. While the relatively high removal rates of pupae in cells treated with (Z)-10-tritriacontene indicates that this or a similar assay may be useful for selection of hygienic honey bees, more studies are needed to determine an optimal dose for triggering uncapping of cells and a) removal of diseased brood or b) recapping of healthy brood.

c. Prophetic Example: Induced Hygienic Behavior Study with P32-Spiked Extracts

Hexane will be used to extract cuticular chemicals from 200 mite-infested and 200 non-infested honey bee larvae aged 4-days post capping. The pooled mite-infested and pooled non-infested larval extracts will each be divided into five vials of 40 brood equivalents (Beq) each, and spiked with 40 μl of one of the following: hexane, 10⁻³ P32, 10⁻⁵ P32, 10⁻⁷ P32, 10⁻⁹ P32. Spiked extracts in each vial will be evaporated under a stream of nitrogen and reconstituted in 40 μl of hexane. At day 4 post-capping, wax-caps of cells and/or larvae in each cell will be treated with 1 μl of each extract type. Forty cells will receive each treatment for a total sample size of 400 (40 cells×2 extract types×5 P32 treatments).

Treatments will be randomly distributed across experimental combs and the location of each treatment recorded on a clear transparent sheet put over the comb. Cell uncapping behavior and removal of brood will be surveyed 1 hour, 4 hours, 8 hours, and 24 hours after treatment. Chi-squared analysis will be used to test for differences in the frequency of hygienic behavior between treatment groups at each time point.

d. Prophetic Example: Commercial Scale Induced Hygienic Behavior Study with P32 Treatment for Varroa-Infested Honey Bee Hives

The P32 compound will be synthesized at a larger scale. Based on the results of the smaller-scale studies above in subsection 4.1.b, an application to deliver the most efficient P32 concentration will be formulated and readily determined by those skilled in the art. The formulation may be a spray that can be applied homogenously onto brood frames.

Thirty experimental hives will be established and integrated into the regular operation of a commercial beekeeper. To simulate the Varroa mite infestation of more mature hives, each experimental hive will receive 20 adult mites, obtained by “sugar shaking” [105]. A comprehensive pre-assessment of the experimental hives, including Varroa mite populations and virus prevalence, will be conducted followed by the actual experiment applying P32 as a Varroa treatment [59, 105, 209]. Specific variables that will be assessed are Varroa mite counts on sticky bottom boards and Varroa population determined by alcohol washes of adult bees [105], the prevalence of ten viruses in 24 individual pupae [210], queen presence and drone population, open and capped brood quantity and solidness, worker population, and stored pollen and honey, according to existing protocols [209]. Throughout the active season until August, P32 treatments will be performed in 15 randomly selective hives, while the remaining 15 will remain untreated controls. P32 will be applied every 21 days, according to the length of one honey bee brood cycle. Although the Varroa life cycle is considerably shorter than this interval, this application frequency will be effective and an even lower frequency is anticipated to be optimal from an economic perspective.

Just prior to each P32 application and at the end of the experimental period, hives will be inspected as above, and the data will be recorded in the field for subsequent analyses by simple comparisons of hive strength and health variables between treatment and control hives. In addition, subsequent overwintering success of these hives will be reported to gain a comprehensive understanding of the impact of a P32 treatment in a commercial setting.

e. Prophetic Example: Bioassay for Hygienic Behavior in Honey Bees

Methods:

The timing of the induced uncapping behavior determined in the experiment of in subsection 4.1.b above will inform this bioassay study. The conventional “freeze-killed brood” assay [100] will be compared to a new P32-spray assay for labor, reproducibility, and predictive power of overall performance outcome of the assayed hives.

A sample of 120 representative hives in different locations will be tested with both assays simultaneously. In parallel to the “freeze-killed brood” assay, P32 will be sprayed on a section of capped brood comb of identical size. Uncapping and removal of brood will be recorded for both assays 2, 4, 8, and 24 hours after treatment. On the following day, both assays will be repeated on a second frame from the same hives. The 16 different variables (4 time points×2 assays×2 outcomes: uncapped or removed) will be assessed for repeatability by correlating the scores from the first day with the corresponding scores from the second day. For each hygienicity variable an overall value will be computed as the average of corresponding scores from both days. The size and health of the 120 colonies will also be assessed [209] at this time. The survival of the hives will be recorded throughout the season and a final assessment of Varroa population [105], size and health will be performed. The relation between hive performance and the overall values of the hygienicity variables will be quantified in a general linear model to assess the predictive power of each variable.

Results:

A trial of this experiment was completed on a small scale. In 10 colonies with various selective breeding histories, removal of brood in capped cells treated with 1% (Z)-10-tritriacontene applied by airbrushing was compared, side-by-side in single frames, to removal of brood in capped cells treated with liquid nitrogen (FKB assay). The frequency of uncapping and removal in each assay was recorded at two hours and twenty-four hours post treatment. Results indicate a significant positive correlation at 24 hours (Pearson Correlation Coefficient=0.688, p=0.014) between uncapping and removal of cells treated with P32 and those treated with liquid nitrogen (FIG. 14).

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All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims. 

What is claimed:
 1. A method of inducing hygienic behavior in honey bees, the method comprising contacting hive cells with a composition comprising a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees.
 2. The method of claim 1, wherein the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof.
 3. The method of claim 1, wherein the tritriacontene is a cis-isomer.
 4. The method of claim 3, wherein the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof.
 5. The method of claim 1, wherein the hygienic behavior comprises eating diseased brood, eating diseased honey bees, removing diseased brood from hive cells, removing diseased honey bees from hive cells, removing pests or parasites, uncapping hive cells, or uncapping and recapping hive cells.
 6. The method of claim 1, wherein the hygienic behavior results in survival of a honey bee colony.
 7. The method of claim 1, wherein the hygienic behavior results in suppression of mite reproduction, decreased mite survival, or suppression of a mite infestation.
 8. The method of claim 1, wherein the hive cells are capped hive cells or uncapped hive cells.
 9. The method of claim 1, wherein the hive cells are worker-brood cells, drone-brood cells, or queen bee cells.
 10. The method of claim 5, wherein the diseased brood are eggs, larvae, or pupae.
 11. The method of claim 5, wherein the diseased brood or diseased honey bees are infested with pests or parasites; infected with a pathogen; or damaged.
 12. The method of claim 5 or 11, wherein the pests or parasites are mites, wax moths, or small hive beetles.
 13. The method of claim 11, wherein the pathogen is a bacterium, a fungus, or a virus.
 14. The method of claim 13, wherein the pathogen causes chalkbrood, American foulbrood, or European foulbrood.
 15. The method of claim 11, wherein the damaged diseased brood or damaged diseased honey bees are health-compromised due to exposure to toxic chemicals.
 16. The method of claim 7 or 12, wherein the mites are mites of the genus Varroa.
 17. The method of claim 16, wherein the mites are mites of the species Varroa destructor or Varroa jacobsoni.
 18. The method of claim 1, wherein the contacting of the hive cells is on one or more days after the hive cells are capped.
 19. The method of claim 1, wherein the contacting of the hive cells is on one or more days before the hive cells are capped.
 20. A method for selecting one or more honey bee(s) exhibiting hygienic behavior comprising: a) applying a tritriacontene composition to a set of hive cells; b) performing an assay to identify a hygienic colony, wherein the assay comprises exposing the set of hive cells to a test colony; and c) selecting one or more honey bee(s) from an identified hygienic colony, wherein the selected one or more honey bee(s) exhibit hygienic behavior.
 21. The method of claim 20, wherein the set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites.
 22. The method of claim 20, wherein the tritriacontene composition comprises a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees.
 23. The composition of claim 22, wherein the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof.
 24. The method of claim 22, wherein the tritriacontene is a cis-isomer.
 25. The method of claim 24, wherein the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof.
 26. The method of claim 20, wherein the selected honey bee(s) is a queen bee or a drone bee.
 27. The method of claim 21, wherein the assay further comprises: i. determining an amount of emptied hive cells in the set of hive cells; and ii. identifying a hygienic colony, wherein a test colony is a hygienic colony if at least 90% of the set of hive cells are emptied.
 28. The method of claim 27, wherein the emptied hive cells have no eggs, larvae, or pupae; or contain partially eaten larvae or pupae.
 29. The method of claim 27, wherein the emptied hive cells are capped hive cells or uncapped hive cells.
 30. The method of claim 20, wherein the hive cells are capped hive cells and the assay further comprises: i. determining the amount of uncapped hive cells in the set of capped hive cells; and ii. identifying a hygienic colony, wherein a test colony is a hygienic colony if at least 90% of the set of capped hive cells are uncapped.
 31. The method of claim 30, wherein the capped hive cells are empty.
 32. The method of claim 20, wherein the hive cells are capped hive cells and the assay further comprises: i. determining the amount of hive cells uncapped and subsequently recapped in the set of capped hive cells; and ii. identifying a hygienic colony, wherein a test colony is a hygienic colony if at least 90% of the set of capped hive cells are uncapped and subsequently recapped.
 33. The method of claim 32, wherein the capped hive cells are empty.
 34. The method of claim 26, further comprising d) mating a selected honey bee with one or more honey bee(s) from at least one separately identified hygienic colony to produce offspring.
 35. The method of claim 26, wherein a queen bee is selected and mated: (a) naturally with one or more drones from at least one separately identified hygienic colony; or (b) artificially inseminated with semen from one or more drones from at least one separately identified hygienic colony.
 36. The method of claim 34, further comprising: e) raising the offspring, f) applying a tritriacontene composition to a second set of hive cells, g) performing a second assay to identify whether the raised offspring is a hygienic colony, wherein the second assay comprises exposing the second set of hive cells to the raised offspring, and h) selecting one or more honey bee(s) from an identified hygienic colony, wherein the selected one or more honey bee(s) exhibit hygienic behavior.
 37. The method of claim 36, wherein the second set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites.
 38. The method of claim 34, wherein at least one separately identified hygienic colony was bred or identified by a method comprising the steps of claim
 15. 39. The method of claim 34, wherein at least one separately identified hygienic colony was bred or identified by a method based on freeze-killed brood, suppression of mite reproduction, or removal of damaged brood.
 40. The method of claim 37, wherein the second assay further comprises: i. determining the amount of emptied hive cells in the second set of hive cells; and ii. identifying a hygienic colony, wherein the raised offspring is a hygienic colony if at least 90% of the second set of hive cells are emptied.
 41. The method of claim 36, wherein the hive cells are capped hive cells and the second assay further comprises: i. determining an amount of uncapped hive cells in the second set of capped hive cells; and ii. identifying a hygienic colony, wherein the raised offspring is a hygienic colony if at least 90% of the second set of capped hive cells are uncapped.
 42. The method of claim 41, wherein the capped hive cells are empty.
 43. The method of claim 36, wherein the hive cells are capped hive cells and the second assay further comprises: i. determining an amount of hive cells uncapped and subsequently recapped in the second set of capped hive cells; and ii. identifying a hygienic colony, wherein the raised offspring is a hygienic colony if at least 90% of the second set of capped hive cells are uncapped and subsequently recapped.
 44. The method of claim 43, wherein the capped hive cells are empty.
 45. A method for assessing the degree of hygienic behavior within a honey bee colony comprising: a applying a tritriacontene composition to a set of hive cells; b. exposing the set of hive cells to a honey bee colony; and c. determining the amount of emptied hive cells in the set of hive cells; wherein a higher amount of the set of hive cells that are emptied is associated with a greater degree of hygienic behavior.
 46. The method of claim 45, wherein the set of hive cells contains a diseased brood, diseased honey bees, or pests or parasites.
 47. The method of claim 45, wherein the tritriacontene composition comprises a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees.
 48. The method of claim 47, wherein the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof.
 49. The method of claim 47, wherein the tritriacontene is a cis-isomer.
 50. The method of claim 49, wherein the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof.
 51. A method for assessing the degree of hygienic behavior within a honey bee colony comprising: a. applying a tritriacontene composition to a set of capped hive cells; b. exposing the set of capped hive cells to a honey bee colony; and c. determining the amount of uncapped hive cells in the set of capped hive cells; wherein a higher amount of the set of hive cells that are uncapped is associated with a greater degree of hygienic behavior.
 52. The method of claim 51, wherein the set of capped hive cells contains a diseased brood, diseased honey bees, or pests or parasites.
 53. The method of claim 51, wherein the set of capped hive cells is empty.
 54. The method of claim 51, wherein the tritriacontene composition comprises a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees.
 55. The method of claim 54, wherein the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof.
 56. The method of claim 54, wherein the tritriacontene is a cis-isomer.
 57. The method of claim 56, wherein the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof.
 58. A method for assessing the degree of hygienic behavior within a hone) bee colony comprising: a. applying a tritriacontene composition to a set of capped hive cells; b. exposing the set of capped hive cells to a honey bee colony; and c. determining the amount of hive cells uncapped and subsequently recapped in the set of capped hive cells; wherein a higher amount of the set of hive cells that are uncapped and subsequently recapped is associated with a greater degree of hygienic behavior.
 59. The method of claim 58, wherein the set of capped hive cells contains a diseased brood, diseased honey bees, or pests or parasites.
 60. The method of claim 58, wherein the set of capped hive cells is empty.
 61. The method of claim 58, wherein the tritriacontene composition comprises a tritriacontene and an agriculturally acceptable diluent or carrier, wherein the tritriacontene is in an effective amount for inducing hygienic behavior in honey bees.
 62. The method of claim 61, wherein the tritriacontene is of the structure:

or agriculturally acceptable derivatives thereof.
 63. The method of claim 61, wherein the tritriacontene is a cis-isomer.
 64. The method of claim 63, wherein the tritriacontene is of the structure:

or an agriculturally acceptable derivative thereof. 